Antigen presenting scaffolds for immune-cell manipulation

ABSTRACT

The present invention relates to artificial antigen presenting cell (aAPC) scaffolds to provide cells with specific functional stimulation to obtain phenotypic and functional properties ideal to mediate tumor regression or viral clearance. In particular, the scaffolds of the present invention comprise antigens, such as peptide-MHC (pMHC) class I molecules, and specific combinations of cytokines and co-stimulatory molecules to allow effective expansion and functional stimulation of specific T cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application Number PCT/EP2017/083862, filed on Dec. 20,2017, designating the United States of America and published in theEnglish language, which is an International Application of and claimsthe benefit of priority to European Patent Application No. 16205918.2,filed on Dec. 21, 2016. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-PLOUG39-082APC.txt, the date of creation of the ASCII text fileis Jun. 14, 2019, and the size of the ASCII text file is 1 KB.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to artificial antigen presenting cell(aAPC) scaffolds to provide cells with specific functional stimulationto obtain phenotypic and functional properties ideal to mediate tumorregression or viral clearance. In particular, the scaffolds of thepresent invention comprise antigens, such as peptide-MHC (pMHC) class Imolecules, and specific combinations of cytokines and co-stimulatorymolecules to allow effective expansion and functional stimulation ofspecific T cells.

BACKGROUND OF THE INVENTION

The immunotherapeutic approach adoptive cell transfer (ACT), in whichtumor-reactive T cells from peripheral blood (PBMC) or tumorinfiltrating lymphocytes (TILs) are extracted from a patient, activatedand expanded ex vivo, and subsequently given back to the patient, has inmalignant melanoma studies showed clinical durable responses in morethan 50% of patients. However, the expansion of tumor-reactive T cellsfrom PBMCs or TILs requires extensive ex vivo culturing often at thecost of T cell differentiation and functional capacity. As a result, thetransferred T cell product may not contain a sufficient frequency oftumor-reactive CD8 T cells with the appropriate phenotypic andfunctional characteristics to mediate tumor regression. Furthermore, themajority of such tumor infiltrating T cells may not be tumor specificbut rather bystander infiltration of T cells from the periphery, with aT cell receptor (TCR) recognition not matching any tumor antigens.Finally, the fraction of tumor-reactive T cells may have a reducedgrowth potential due to the suppressive environment present at the tumorsite.

Attempts have been made to utilize artificial antigen presenting cells(aAPCs) to overcome the problem of insufficient differentiation andfunctional capacity of the expanded T cells. The simple concept behindaAPCs is that they mimic the natural interaction between the TCR and thespecific peptide antigen presented by the major histocompatibilitycomplex (MHC). This interaction is the core step in generation ofimmunity through activation, expansion and differentiation of T cellsthat are capable of eliciting an efficient immune response. The naturalgeneration of a T cell response is further aided by cytokines andco-stimulatory molecules, which serves to induce T cell activation andfunction. Thus, incorporation of all the necessary molecules into asingle aAPC scaffold is a promising tool to overcome some of thechallenges of expansion of T cells. The aAPCs form the idealimmunological synapse for T cell activation and differentiation.However, a crucial challenge is the uncovering of combinations ofmolecules enabling the aAPCs to efficiently expand the extracted TILswhile also maintaining a functional phenotype.

In WO2002072631 are disclosed many concepts of utilizing MHC platforms,wherein one of them is a MHC construct comprising a carrier moleculehaving attached thereto one or more MHC molecules. The construct mayalso contain biologically active molecules such as co-stimulatorymolecules or cell modulating molecules. The MHC construct is envisionedamongst others to be used for expansion of cells recognizing theconstruct and used to generate a therapeutic composition for use intreatment of disease, such as cancer and others. WO2002072631 disclosesmany co-stimulatory molecules and cytokines that may be suitable for Tcell expansion, but fails to identify any specific combinationsparticularly suitable and effective for the purpose of expansion of Tcells.

US 2011/318380 disclose application of the MHC construct described inWO2002072631 for cancer vaccines and immune monitoring. However, US2011/318380 do not exemplify any specific combinations of co-stimulatorymolecules and cytokines particularly suitable and effective for thepurpose of expansion of T cells.

WO2009003492 is mainly focused on detection of antigen specific T cells,but also discloses the expansion of antigen specific T cells. Describedtherein are MHC multimers with and without complexed peptides, methodsfor their preparation and methods for their use in analysis and therapy,including isolation of antigen specific T-cells capable of inactivationor elimination of undesirable target T-cells. The MHC multimersaccording to WO2009003492 may comprise a dextran scaffold andco-stimulatory- and cell modulating molecules. However, the disclosurefails to pinpoint specific combinations of molecules especiallyeffective for the purpose of expansion of T cells.

In WO2009094273 is disclosed an aAPC composition comprisingnanoparticles, cytokines, coupling agents, T cell receptor activatorsand co-stimulatory molecules for use to expand antigen-specific T cells.The T cell receptor activator may be an MHC molecule bound to a peptideantigen. Furthermore, the use of the expanded T cells in adoptiveimmunotherapy is described. However, only the suitability of a singlecytokine on an aAPC, namely IL-2, is explored and only in comparisonwith the exogenous cytokine.

Thus, common for the previous disclosures of aAPC scaffolds is that theyonly describe the concept in a largely generic manner. Since the successcriteria for T cell expansion, i.e. high ratio of active T cells, highantigen specificity of the T cells and high functionality of the Tcells, is only met when specific combinations of stimulatory moleculesare combined, a great need for well-defined and effective aAPC scaffoldsexists. Only when all of the three success criteria for T cell expansionis fulfilled will the resulting population of T cells be optimallyprepared to apply their antitumor or antiviral functions.

Hence, improved aAPC scaffolds would be advantageous. In particular, theprovision of more efficient aAPC scaffolds with high ratio of active Tcells, high antigen specificity of the T cells and high functionality ofthe T cells would be in demand.

SUMMARY OF THE INVENTION

Thus, an object of the present invention relates to the provision ofartificial antigen presenting cell (aAPC) scaffolds with improvedcapabilities for expansion of tumor-reactive T cells extracted fromperipheral blood (PBMC) or tumor infiltrating lymphocytes (TILs).

In particular, it is an object of the present invention to provide anaAPC scaffold that solves the above mentioned problems of the prior artof insufficient T cell differentiation and functional capacities of theexpanded T cell population.

Another object of the present invention is to utilize the obtainedexpanded T cell populations with optimized phenotypic and functionalproperties to mediate tumor regression or viral clearance.

Thus, one aspect of the invention relates to an artificial antigenpresenting cell (aAPC) scaffold comprising a polymeric backbone to whichare attached the following template molecules:

-   -   i. at least one major histocompatibility complex molecule        comprising an antigenic peptide (pMHC),    -   ii. at least one cytokine selected from the group consisting of        IL-21, IL-2, IL-15, IL-1, IL-6, IL-10 and IL-7,    -   iii. optionally, at least one co-stimulatory molecule selected        from the group consisting of B7.2 (CD86), B7.1 (CD80), CD40,        ICOS and PD-L1, and    -   iv. optionally, at least one CD47 molecule.

A preferred aspect of the invention relates to an artificial antigenpresenting cell (aAPC) scaffold comprising a polymeric backbone to whichare attached the following template molecules:

-   -   i. at least two different gamma-chain receptor cytokines, such        as at least two different gamma-chain receptor cytokines        selected from the group consisting of IL-21, IL-2, IL-15, IL-4,        IL-9 and IL-7,    -   ii. at least one antigen,    -   iii. optionally, at least one co-stimulatory molecule selected        from the group consisting of B7.2 (CD86), B7.1 (CD80), CD40,        ICOS and PD-L1, and    -   iv. optionally, at least one CD47 molecule.

Another aspect of the present invention relates to a method forsimultaneous in vitro stimulation and expansion of T cells, comprisingthe following steps:

-   -   i. providing a sample comprising T cells,    -   ii. contacting said sample with a solution comprising an aAPC        scaffold according to the present invention,    -   iii. stimulating and expanding T cells with specificity for said        aAPC scaffold in culture, and    -   iv. harvesting the T cells of step iii) from the culture to        obtain an expanded antigen-specific population of T cells.

A further aspect of the present invention is to provide an expanded Tcell population obtained by the method according to the presentinvention.

Yet another aspect of the present invention relates to an expandedT-cell population obtained by the method according to present inventionfor use as a medicament.

Still another aspect of the present invention is to provide an expandedT-cell population obtained by the method according to the presentinvention for use in the treatment of a cancer or viral condition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows (A) a schematic overview over an exemplary artificialantigen presenting cell (aAPC) scaffold. The aAPC scaffold is comprisedof a backbone to which are attached template molecules such aspeptide-MHC (pMHC) molecules, cytokines and optionally co-stimulatorymolecules. Furthermore, CD47 molecules may be attached to the aAPCscaffold. Examples are given of aAPC scaffolds, wherein different ratiosof the backbone and template molecules are assembled into aAPCscaffolds. (B) Illustration of how carefully selected combinations oftemplate molecules may be combined in an aAPC scaffold and utilized toexpand specific T cell populations extracted from patients.

FIG. 2 shows (A) Mean fluorescent intensity (MFI) values for T cellsstained using different antigen presenting scaffolds assembled inscaffold:pMHC ratios of 1:1, 1:5, 1:10, 1:20, 1:30 and applied instaining of PBMCs from a healthy donor with response against CMV pp65YSE peptide. (B) MFI value and (C) SI values for T cell samples stainedusing different antigen presenting scaffolds assembled in scaffold:pMHCratios of 1:10 or 1:20 and co-attached with B7-2 and IL-15 asco-stimulatory molecules in a ratio of 5:5.

FIG. 3 shows scaffolds assembled with either (A) B7-2 or (B) IL-15 inratio 1:30 and applied in staining of healthy donor PBMCs. Fluorochromeon Y-axis is PE-Cy7 and flourochrome on X-axis is FITC.

FIG. 4 shows (A) Frequency of HLA-A1 FLU BP-VSD specific CD8 T cellsfrom a healthy donor detected directly ex vivo with PE (X-axis) and APC(Y-axis) labeled tetramers. (B) Frequency of HLA-A1 FLU BP-VSD specificCD8 T cells after two weeks culturing with antigen presenting scaffoldswith either the ratio 1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21), plus20 IU/ml IL-2 added in the culture media, (C) free FLU BP-VSD peptide,IL-15, and IL-21, or (D) Antigen presenting scaffold with the ratio1:10:5:5:5 carrying an irrelevant peptide specificity. (E) Expansionrate based on frequency of HLA-A1 FLU BP-VSD specific CD8 T cells,detected by tetramer staining from baseline, 1 week and 2 weeks afterexpansion. (F) Absolute number of HLA-A1 FLU BP-VSD specific CD8 T cellsafter 2 weeks expansion.

FIG. 5 shows (A) Frequency of HLA-A1 FLU BP-VSD specific CD8 T cellsfrom a healthy donor detected directly ex vivo with PE (X-axis) and APC(Y-axis) labeled tetramers. (B) Frequency of HLA-A1 FLU BP-VSD specificCD8 T cells after two weeks culturing with either filtered antigenpresenting-scaffolds with the ratio 1:15:5:5 (scaffold:pMHC:B7-2:IL-15),unfiltered antigen presenting scaffolds or free FLU BP-VSD peptide inthe culture media, plus 20 IU/ml IL-2 was added to all cultures. Thefrequencies were detected with APC and PE labeled tetramers. (C)Expansion rate based on frequency of HLA-A1 FLU BP-VSD specific CD8 Tcells, detected by tetramer staining from baseline, 1 week and 2 weeksafter expansion. (D) MFI values from tetramer positive CD3/CD8 T cellsafter 2 weeks expansion with either filtered antigen presentingscaffolds with the ratio 1:15:5:5 (scaffold:pMHC:B7-2:IL-15), unfilteredantigen presenting scaffolds or free FLU BP-VSD peptide.

FIG. 6 shows (A) Frequencies of HLA-A3 FLU NP LIR specific CD8 T cellsfrom a healthy donor after 2 weeks expansion with either unfiltered orfiltered antigen presenting scaffold with the ratio 1:15:5:5(scaffold:pMHC:B7-2:IL-15). The dot plots show two populations ofantigen-specific CD8 T cells, one binding with high affinity (blackpopulation) and another binding with lower affinity (dark greypopulation) to PE-Cy7 labeled tetramers (X-axis), while equal stainingintensity is obtained from the CD8 antibody, PerCP labeled (Y-axis). (B)Bar chart of CD28 expression of HLA-A3 FLU NP LIR specific CD8 T cellswith high and low binding affinity to tetramers after 2 weeks expansionwith either unfiltered or filtered antigen presenting scaffold with theratio 1:15:5:5.

FIG. 7 shows (A) Frequency of HLA-A1 FLU BP-VSD specific CD8 T cellsfrom a healthy donor detected by tetramer staining after 2 weeksstimulation either with unfiltered or filtered antigen presentingscaffold with the ratio 1:15:5:5 (scaffold:pMHC:B7-2:IL-15). CD8antibody is PerCP labeled (Y-axis) and the tetramer is PE-Cy7 labeled(X-axis). (B) Dot plots showing frequency of CD45RA and CD28 expression,(C) CD45RA and CCR7 expression, and (D) CD45RA and CD57 expression ofHLA-A1 FLU BP-VSD specific CD8 T cells after 2 weeks expansion witheither unfiltered or filtered antigen presenting scaffold with the ratio1:15:5:5.

FIG. 8 shows expression of CD28, CD57 and CCR7 of HLA-A1 FLU BP-VSDspecific CD8 T cells after 2 weeks expansion with (A) filtered andunfiltered antigen presenting scaffold with ratio 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21) compared with free peptide IL-15 andIL-21, all these cultures had 20 IU/ML IL-2 in the culture media. (B)Filtered and unfiltered antigen presenting scaffold with ratio 1:8:8:8(scaffold:pMHC:IL-2:IL-21) compared with antigen presenting scaffold 1:8(scaffold:pMHC) with free IL-2 and IL21. (C) MFI value of CD28expression from HLA-A1 FLU BP-VSD specific CD8 T cells after 2 weeksexpansion with filtered and unfiltered antigen presenting scaffold withratio 1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21) or free peptide, IL-15and IL-21. (D) MFI value of CD28 expression from HLA-A1 FLU BP-VSDspecific CD8 T cells after 2 weeks expansion with filtered andunfiltered antigen presenting scaffold with ratio 1:8:8:8(scaffold:pMHC:IL-2:IL-21) or antigen presenting scaffold 1:8(scaffold:pMHC) with free IL-2 and IL21.

FIG. 9 shows expression of PD-1, Tim-3, and LAG-3 of HLA-A1 FLU BP-VSDspecific CD8 T cells after 2 weeks expansion with (A) filtered andunfiltered antigen presenting scaffold with ratio 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21) compared with free peptide, IL-15 andIL-21, all these cultures had 20 IU/ML IL-2 in the culture media. (B)Filtered and unfiltered antigen presenting scaffold with ratio 1:8:8:8(scaffold:pMHC:IL-2:IL-21) compared with antigen presenting scaffold 1:8(scaffold:pMHC) with free IL-2 and IL21. (C) Frequency of PD-1 negativeHLA-A1 FLU BP-VSD specific CD8 T cells after 2 weeks expansion withfiltered and unfiltered antigen presenting scaffold with ratio1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21) or free peptide, IL-15 andIL-21. (D) Frequency of PD-1 negative HLA-A1 FLU BP-VSD specific CD8 Tcells after 2 weeks expansion with filtered and unfiltered antigenpresenting scaffold with ratio 1:8:8:8 (scaffold:pMHC:IL-2:IL-21) orantigen presenting scaffold 1:8 (scaffold:pMHC) with free IL-2 and IL21.

FIG. 10 shows frequency of TNF-α, IFN-γ and CD107a expression after invitro peptide stimulation using HLA-A1 FLU BP-VSD peptide. Cytokinesecretion in peptide responsive CD8 T cells was measured byintracellular cytokine staining. The tested cell cultures were obtainedafter 2 weeks expansion with either (A) filtered and unfiltered antigenpresenting scaffolds of ratio 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21) compared with free peptide and IL-15,IL-21 stimulation, or (B) filtered and unfiltered antigen presentingscaffolds of ratio 1:8:8:8 (scaffold:pMHC:IL-2:IL-21) with no IL-2 inthe culture media, compared with antigen presenting scaffold 1:8(scaffold:pMHC) with free IL-2 and IL-21. The diagrams show thefrequency of triple, double and single positive HLA-A1 FLU BP-VSDspecific CD8 T cells. The triple positive fraction is highlighted(through elevation) in each diagram.

FIG. 11 shows dot plots showing the expression of (A) CD107a and IFN-γ,and (B) TNF-α and IFN-γ among CD8 T cells following stimulation withHLA-A1 FLU BP-VSD peptide. Cultures were stimulated for 2 weeks withantigen presenting scaffolds with ratio 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21) carrying either relevant (left plots)or irrelevant peptide specificity (right plots) in the MHC complex. In(A) the CD107a antibody is PE labeled (Y-axis) and the IFN-γ antibody isAPC labeled (X-axis), in (B) the TNF-α antibody is PE-Cy7 labeled(Y-axis) and the IFN-γ antibody is APC labeled (X-axis). These stainingswere made in duplicate, only one of each staining is shown.

FIG. 12 shows fold expansion of 4 virus responses from a healthy donorafter 2 weeks expansion with antigen presenting scaffolds with the ratio1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21). Five cultures wereestablished:

1-4. 4 virus responses were expanded in individual cultures (one perculture)

5. The 4 virus responses were expanded simultaneously (4 per culture)

The specificity of the 4 evaluated peptide-MHC responses were: HLA-A2FLU MP 58-66 GIL, HLA-A2 EBV LMP2 FLY, HLA-A2 CMV pp65 NLV and HLA-A2EBV BRLF1 YVL.

FIG. 13 shows (A) Expression of TNF-α, IFN-γ and CD107a within T cellcultures stimulated with the respective antigen presenting scaffolds for2 weeks, and thereafter exposed to the specific peptide (HLA-A2 EBV LMP2CLG) for 4 hours. 21 different antigen presenting scaffolds with ratio1:10:5:5:5 (scaffold:pMHC:B7-2:molecule 1:molecule 2) were used. Theoutermost black circle represents the absolute number ofantigen-specific CD8 T cells (events) detected with tetramer staining,circle 1 refers to expression of one of the three markers (TNF-α, IFN-γand CD107a), circle 2 refers to expression of the two of the threemarkers, and circle 3 refers to expression of all three markers. (B)Reference antigen presenting scaffold with the ratio 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21).

FIG. 14 shows (A) Expression of TNF-α, IFN-γ and CD107a within T cellcultures stimulated with the respective antigen presenting scaffolds for2 weeks, and thereafter exposed to the specific peptide (HLA-A2 EBV LMP2CLG) for 4 hours. 7 different antigen presenting scaffolds with ratio1:8:8:8 (scaffold:pMHC:IL-2:molecules 1) were used. Molecule 1 variedbetween PD-L1, ICOS, OX40L, CD5, IL-1 IL-6, IL-10. The outermost blackcircle represents the absolute number of antigen-specific CD8 T cells(events) detected with tetramer staining, circle 1 refers to expressionof one of the three markers (TNF-α, IFN-γ and CD107a), circle 2 refersto expression of the two of the three markers, and circle 3 refers toexpression of all three markers. (B) Reference antigen presentingscaffold with the ratio 1:8:8:8 (scaffold:pMHC:IL-2:IL-21).

FIG. 15 shows CD28 and PD-1 expression of HLA-A2 EBV LMP2 CLG specificCD8 T cells after 2 weeks expansion with (A) 19 different antigenpresenting scaffolds with ratio 1:10:5:5:5 (scaffold:pMHC:B7-2:molecule1:molecule 2) and a reference antigen presenting scaffold(scaffold:pMHC:B7-2:IL-15:IL-21), and (B) seven antigen presentingscaffold with ratio 1:8:8:8 (scaffold:pMHC:IL-2:molecules 1) and areference antigen presenting scaffold (scaffold:pMHC:IL-2:IL-21) withoutIL-2 in the culture media. The black circle represents the absolutenumber of antigen-specific CD8 T cells (events) detected with tetramerstaining, the relative distribution on the x- and y-axes representstheir expression of the two molecules, PD-1 and CD28. PD-1 and CD28antibodies were both BV-421 labeled.

FIG. 16 shows frequency of HLA-A2 EBV LMP2 CLG specific CD8 T cells froma healthy donor detected by tetramer staining after two weeksstimulation with antigen presenting scaffolds, assembled with differentscaffold length of 270 kDa from Immudex, and 250 kDa, 750 kDa, and 2000kDa from Fina Biosolutions. The scaffolds were assembled with the ratio1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21). CD8 antibody is BV510labeled (Y-axis) and the tetramer is PE labeled (X-axis). The baselineresponse of the HLA-A2 EBV LMP2 CLG specific CD8 T cells was 0.01%.

FIG. 17 shows parallel expansion of antigen specific CD8 T cells from ahealthy donor, with five different known virus responses using aAPCscaffold 1:8:8:8 (scaffold:pMHC:IL-2:IL-21). (A) Two of the fivedifferent virus responses (HLA-A2 EBV LMP2 FLY and HLA-A2 CMV pp65 NLVspecific CD8 T cells) were respectively expanded either individually oras a mixture of 1/10 the specific aAPC scaffold plus 9/10 of aAPCscaffold with irrelevant non-matching HLA-types. (B) All five virusresponses were expanded simultaneously in the same culture, using 1/10of the normal aAPC scaffold concentration for each specificity, plus5/10 of aAPC scaffolds with non-matching HLA-type. The specificity ofthe five virus responses are respectively, HLA-A2 FLU MP 58-66 GIL,HLA-A2 EBV LMP2 FLY, HLA-A2 CMV pp65 NLV, HLA-A2 EBV BRLF1 YVL, andHLA-A2 CMV IE1 VLE.

FIG. 18 shows the frequency of antigen-specific CD8 T cells from ahealthy donor after 2 weeks expansion with aAPC scaffolds of 250 KDa,750 KDa, and 2000 KDa. Two different scaffold to molecule ratios wereused for all three scaffolds, (A) aAPC scaffold 1:8:8:8(scaffold:pMHC:IL-2:IL-21), and (B) aAPC scaffold 1:24:24:24(scaffold:pMHC:IL-2:IL-21).

FIG. 19 shows aAPC scaffold-mediated in vivo expansion of OVA-specificCD8 T cells in C57BL/6 mice. Frequency of OVA-specific CD8 T cells weremeasured pre vaccination, on day 7 and day 19 after i.p. vaccinationwith OVA+poly IC and day 7 after booster. Four different boosters wereadministrated on day 21 post vaccination. Mouse 1 had PBS i.v., mouse 2had OVA i.p., mouse 3 had aAPC scaffold 1:8:8:8 with the H2-Kb/SIINFEKL(scaffold:pMHC:IL-2:IL21) i.v., and mouse 4 had H2-Kb/SIINFEKL in thesame concentration as assembled on the aAPC scaffold 1:8:8:8 i.v. (i.e.the booster for mouse 3). I.e. in mouse 4, the antigenic peptide wasgiven as part of a pMHC complex, but without the aAPC scaffold.

FIG. 20 shows a comparison of aAPC scaffold-mediated expansion versusmonocyte-derived dendritic cells (moDC)-mediated expansion ofantigen-specific T cells. Antigen-specific T cells were expanded from ahealthy donor with initially 0.01% antigen-specific T cells. Theexpansion was done under four conditions in parallel in the presence ofeither (A) free pMHC complex and IL2 and IL21 (i.e. without the aAPCscaffold), (B) aAPC scaffold ratio 1:8:8:8 (scaffold:pMHC:IL-2:IL-21),(C) unpulsed moDC's or (D) peptide pulsed moDC's derived from the same.Antigen-specific T cells subjected to either of the four conditions werecultured for 2 weeks, stimulated as indicated twice a week. Theexpansion of antigen-specific T cells was traced by MHC tetramerstaining after two weeks. Representative dot plots are shown in FIG. 20.

The present invention will now be described in more detail in thefollowing.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Prior to discussing the present invention in further detail, thefollowing terms and conventions will first be defined:

Artificial Antigen Presenting Cell (aAPC) Scaffold

In the present context, the term “artificial antigen presenting cell(aAPC) scaffold” means an assembly of the necessary molecules as definedherein to function similar to an antigen presenting cell.

Polymeric Backbone

In the present context, the term “polymeric backbone” means the part ofthe aAPC scaffold onto which the individual template molecules arefixed. The template molecules are attached by means of an interactionbetween a coupling agent located on or as an integrated part of thepolymeric backbone and an affinity tag placed on the template molecule.Alternatively, the coupling agent may be on the template molecule, withthe corresponding affinity tag being on the polymeric backbone.

The polymeric backbone may be of a material selected frompolysaccharides, vinyl polymers, poly ethylene glycol, poly propyleneglycol, strep-tactin, poly-streptavidin, biotin-binding proteins andpolyhistidine-binding polymers.

Template Molecules

In the present context, the term “template molecule” refers to anymolecule attached onto the polymeric backbone of the aAPC scaffold. Theymay be selected from pMHC molecules, cytokines, co-stimulatory moleculesand CD47. Template molecules comprise an affinity tag.

Non-Covalent Interaction

In the present context, the term “non-covalent interaction” means anybonding via other interactions than a covalent bond. A non-covalent bondmay be formed by e.g. hydrophobic interactions, hydrophilicinteractions, ionic interactions, van der walls forces, hydrogenbonding, and combinations thereof.

Coupling Agent

In the present context, the term “coupling agent” refers to a molecularentity positioned on the polymeric backbone of the aAPC. A couplingagent can be non-covalently bound to an affinity tag. Examples ofcoupling agents include streptavidin, avidin, strep-tactin, antibodies,poly His-tags, metal ion chelates etc.

Alternatively, the coupling agent may be on the template molecule, withthe corresponding affinity tag being on the polymeric backbone.

Affinity Tag

In the present context, the term “affinity tag” refers to a molecularspecies located on a template molecule. An affinity tag binds highlyspecifically to a coupling agent by non-covalent interaction. Examplesof coupling agents include biotin, antibody epitopes, His-tags,streptavidin, strep-tactin, polyhistidine, peptides, metal ion chelatesetc.

Alternatively, the affinity tag may be on the polymeric backbone, withthe corresponding coupling agent being on backbone the templatemolecule.

Antigen

In the present context, the term “antigen” refers to a molecule that iscapable of inducing an immune response, either by itself or inco-operation with other molecules.

The aAPC as defined herein comprises at least one antigen. Theantigen(s) are part of the aAPC, either as i) independent molecules orii) as part of a complex of molecules. In the case of i), the antigenmay be a protein, such as a cluster of differentiation (CD) protein. Inthe case of ii), the antigen may be a protein or in the form of anantigenic peptide. Such antigenic peptide may be part of a complex withthe major histocompatibility complex (MHC), namely a pMHC complex.

The antigen may be a MHC presented antigenic peptide or an antigen thatis recognized without being bound to the MHC complex (i.e. non-MHCpresented molecule). Examples of antigens not presented in complex withMHC include, but are not limited to, CD proteins, such as CD19, CD20 andCD22.

Haptens

In the present context, the term “haptens” refers to small moleculesthat can elicit an immune response only when attached to a largecarrier, such as a protein or a scaffold. Thus, haptens are lowmolecular weight and non-immunogenic compounds that may be bound byantibodies, but do not elicit an immune response on its own. Haptens maybe conjugated to a disease-targeted antibody.

Examples of haptens include, but are not limited to, biotin,fluorescein, digoxigenin, dinitrophenol, cotinine, hydralazine andurushiol.

MHC and pMHC

In the present context, the terms “MHC” and “pMHC” are usedinterchangeably and refer to major histocompatibility complex (MHC)molecules with an antigenic peptide complexed.

In humans, the MHC complex is encoded by the human leukocyte antigen(HLA) gene complex. Thus, in the present context, the term “MHC”encompass also “HLA”.

Cytokine

In the present context, the term “cytokine” means an immune-regulatorymolecule that affects expansion, survival and effector function ofstimulated T cells. Cytokines include chemokines, interferons,interleukins, lymphokines, and tumor necrosis factors.

Gamma-Chain Receptor Cytokines

In the present context, the term “gamma-chain receptor cytokines” refersto the group of cytokines that bind to a corresponding cytokine receptorcomprising the common gamma-chain subunit. The common gamma-chain(γ_(c)) receptor is also known as CD132 or interleukin-2 receptorsubunit gamma (IL-2RG). One common denominator for the gamma-chainreceptor cytokines is that they all deliver their intracellular signalthrough the shared gamma-chain receptor and influence T-cell activationand differentiation.

The γ_(c) glycoprotein is a transmembrane protein, which comprisesextracellular, transmembrane and intracellular domains and is typicallyexpressed on lymphocytes. The γ_(c) subunit is part of the receptorcomplexes of at least six different cytokine receptors, namely the IL-2,IL-4, IL-7, IL-9, IL-15 and IL-21 receptors. Therefore, the group ofgamma-chain receptor cytokines comprises at least IL-2, IL-4, IL-7,IL-9, IL-15 and IL-21.

Co-Stimulatory Molecule

In the present context, the term “co-stimulatory molecule” means amolecule that upon interaction with T cells enhances T cell response,proliferation, production and/or secretion of cytokines, stimulatesdifferentiation and effector functions of T cells or promotes survivalof T cells relative to T cells not contacted with a co-stimulatorymolecule. Examples of co-stimulatory molecules include 67.1, B7.2, ICOS,PD-L1, a-galactosylceramide etc.

Epitope

In the present context, the term “epitope” means the antigenicdeterminant recognized by the TCR of the T cell. The epitope presentedby the pMHC is highly specific for any foreign substance and theinteraction with the TCR ensures effective expansion and functionalstimulation of the specific T cells in a peptide-MHC-directed fashion.

Pharmaceutical Composition

In the present context, the term “pharmaceutical composition” refers toa composition comprising an expanded T cell population obtainedaccording to the invention, suspended in a suitable amount of apharmaceutical acceptable diluent or excipient and/or a pharmaceuticallyacceptable carrier.

Pharmaceutically Acceptable

In the present context, the term “pharmaceutically acceptable” refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce an allergic or similar untoward reaction,such as gastric upset, dizziness and the like, when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals, and more particularly inhumans.

Adjuvant

In the present context, the term “adjuvant” refers to a compound ormixture that enhances the immune response to an antigen. An adjuvant canserve as a tissue depot that slowly releases the antigen and as alymphoid system activator, which non-specifically enhances the immuneresponse. Often, a primary challenge with an antigen alone, in theabsence of an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Excipient

In the present context, the term “excipient” refers to a diluent,adjuvant, carrier, or vehicle with which the composition of theinvention is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or aqueous solution saline solutionsand aqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin.

Artificial Antigen Presenting Cell (aAPC) Scaffold

T cells play a crucial role in the immune response, where they recognizeand respond to foreign substances by interacting with antigen presentingcells (APC), displaying antigenic peptides of the foreign substance incomplex with MHC molecules (pMHC). The T cells are very specific andexpress only a single specificity of T cell receptor (TCR), therebyallowing the T cell only to recognize and respond to a single specificpMHC molecule. When the T cells are first primed to develop receptors ofa specific combination of antigen and MHC molecule, they will notsubsequently be able to recognize other specificities. Thisspecialization of the T cell is called MHC restriction and can beutilized to expand T cells of a single specificity without anyirrelevant specificities “polluting” the expanded T cell population.

Some gene-modified immune cells, such as CAR T cells, recognize antigensthat are not presented by MHC molecules. The present invention may beutilized for expansion of cells recognizing any type of antigen.

Thus, the aAPC may comprise any antigen that is capable of inducing animmune response, either by itself or in co-operation with othermolecules. Such an antigen may be a protein, such as a cluster ofdifferentiation (CD) protein.

MHC molecules exist in several variants, of which MHC class I and MHCclass II molecules may be regarded as the most important. The MHC classI molecules interact with CD8 positive cytotoxic T cells (CD8+ T cells)and MHC class II molecules interact with CD4 positive helper T cells(CD4+ T cells). Once activated CD8+ T cells generally seek to killcancer cells, cells that are infected (particularly with viruses), orcells that are damaged in other ways. CD4+ T cells on the other handmainly function by assisting the immune system, e.g. by releasingcytokines and potentiate the CD8 T cells. Although not limited to asingle type of T cell, the present invention is mainly concerned withthe activation, stimulation and expansion of CD8+ T cells. This isparticularly true since the utilization of an aAPC scaffold, to someextent, fulfills the role of the CD4+ T cells. However, the aAPCscaffolds as described herein may be utilized to expand both CD4+ Tcells and CD8+ T cells.

Although the TCR-pMHC interaction is the main driver for the activationof T cells, several other stimuli are required to prepare the T cellsfor an effective immune response. Overall, the activation of CD8+ Tcells requires two signals; 1) the interaction between the TCR and thepMHC class I molecule and 2) a co-stimulatory interaction between CD28,a membrane receptor on T-cells, and CD28 ligands located on the APC,such as B7.1 (CD80) or B7.2 (CD86). The second signal serves to enhanceproliferation, cytokine production and cell survival.

In addition to the stimulatory signals, T cell response is alsoregulated by inhibitory signals. Tim-3, LAG-3 and PD-1 are examples ofmediators of inhibitory signals. They serve as a natural mechanism toavoid excessive T cell activation and prevent the immune system fromrunning rampant across the organism.

The secondary signal may be assisted, or in some cases replaced, bystimulation of the CD8+ T cell with cytokines released by CD4+ T cells.Thus, cytokines constitute another important group of molecules involvedin the modulation of the immune response. Cytokines generally includeinterleukins, interferons, chemokines, lymphokines, and tumor necrosisfactors. They act through receptors and amongst others regulate thematuration, growth, and responsiveness of T cell populations. Together,interleukin-2 (IL-2) and the co-stimulatory signals are the most crucialfactors for preservation of continuous cell division. The delicateinterplay between co-stimulatory molecules and cytokines is complex andone of the key factors of efficient and specific T cell expansion.

Another molecule that plays a key role in immune responses as well as incellular processes, such as apoptosis, proliferation, adhesion, andmigration, is CD47. This transmembrane protein is ubiquitously expressedin human cells, but is also overexpressed in many different tumor cells,with high levels of CD47 allowing the cancer cells to avoidphagocytosis. However, CD47 is also widely expressed in immune cells,functioning as a “don't eat me” signal that prolongs the circulationtime of the immune cells. Expansion of T cells that express CD47 may bepreferable as these cells are forecasted to have an increased half-lifewhen used therapeutically.

Therefore, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the template molecules comprise aligand capable of stimulating CD47 expression in a T cell population.

CD47 may also infer beneficial properties to the aAPC itself, e.g. as a“don't eat me” signal that prolongs the half-life of the aAPC scaffoldin culture or in circulation.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the template molecules comprise atleast one CD47 molecule.

As exemplified by the above description, there are many factors involvedin the activation and proliferation of T cells. However, for the purposeof immune therapy and/or expansion of a specific T cell population, itis possible to set some conditions that should ideally be fulfilled forthe ability to provide a T cell population with high activity andfunctionality suited for these purposes. Thus, preferablecharacteristics of the expanded T cells include:

-   -   a. high expression of activators (such as CD28)    -   b. low expression of inhibitors (such as PD1)    -   c. a multifunctional cytokine response

The cluster of different molecules required for efficient activation andstimulation has to be present simultaneously to provide the optimalcapacity for T cell function and expansion. The use of an aAPC scaffoldcollects the combination of required molecules in a defined proximity toeach other and thus constitutes a suitable platform for efficientexpansion of the specific T cells.

Thus, the present invention demonstrates specific conditions required toexpand tumor-reactive T cells, through use of MHC-loaded aAPC scaffoldsto provide the cells with specific functional stimulation to obtainphenotypic and functional properties ideal to mediate tumor regressionor viral clearance. These aAPC scaffolds are constructed from apolymeric backbone conjugated with coupling agents to which affinitytagged peptide-MHC (pMHC) molecules are attached to govern the specificinteraction with a specific T cell, and a combination of likewiseaffinity tagged cytokines and co-stimulatory molecules are co-attachedto provide stimulation of the specific T cells to achieve increasedfunctional properties. The aAPC scaffolds will specifically interactwith T cells based on recognition of the pMHC molecule, and can throughthis specific interaction effectively expand and functionally stimulatespecific T cells in a peptide-MHC-directed fashion.

The aAPC scaffolds may be assembled by combinations of a large varietyof different template molecules (i.e. pMHC molecules, cytokines andco-stimulatory molecules). The aAPC scaffolds described herein maycomprise one or more co-stimulatory molecules including, but not limitedto, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD27, CD28, CD30, CD40, CD48,CD58, CD69, CD70, CD72, B7.1 (CD80), CD83, B7.2 (CD86), Fas (CD95), OX40(CD134), CD137 (4-1BB), CD147, SLAM (CDw150), CTLA-4 (CD152), CD153(CD30L), CD40L (CD154), inducible T-cell co-stimulator (ICOS, CD278),CD134L, CD137L, OX40L, NKG2D, HVEM, PD-1, B7RP-1, PD-L1, PD-L2,intercellular adhesion molecule (ICAM) and ICOSL.

Furthermore, the aAPC scaffolds described herein may comprise one ormore cytokines including, but not limited to interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7),interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12),interleukin-15 (IL-15), interleukin-21 (IL-21), interferon alpha(IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), IGIF,granulocyte macrophage colony stimulating factor (GM-CSF), tumornecrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β) andmacrophage colony stimulating factor (M-CSF), and variants and fragmentsthereof.

Herein are described aAPC scaffolds suitable for T cell expansion,ensuring a high ratio of active T cells, high antigen specificity of theT cells and high functionality of the T cells. Consequently, a firstaspect of the present invention relates to an artificial antigenpresenting cell (aAPC) scaffold comprising a polymeric backbone to whichare attached the following template molecules:

-   -   i. at least one major histocompatibility complex molecule        comprising an antigenic peptide (pMHC),    -   ii. at least one cytokine selected from the group consisting of        IL-21, IL-2, IL-15, IL-1, IL-6, IL-10 and IL-7,    -   iii. optionally, at least one co-stimulatory molecule selected        from the group consisting of B7.2 (CD86), B7.1 (CD80), CD40,        ICOS and PD-L1, and    -   iv. optionally, at least one CD47 molecule.

The expansion of some T cells may be enhanced when several cytokines arepresent simultaneously. Thus, an embodiment of the present inventionrelates to the aAPC scaffold as described herein, wherein the templatemolecules comprise at least two different cytokines selected from thegroup consisting of IL-21, IL-2, IL-15, IL-1, IL-6, IL-10 and IL-7.

The aAPC according to the present invention may also comprise antigensthat are recognized without being bound to the MHC complex. Suchantigens may be, but are not limited to, proteins belonging to thecluster of differentiation (CD) classification.

Different groups of cytokines have been identified to produce especiallyfavorable aAPC scaffolds. Without being bound by theory, one efficientgroup of cytokines are cytokines that deliver their intracellular signalthrough the shared gamma-chain receptor and influence T-cell activationand differentiation. In the present context, these cytokines are termed“gamma-chain receptor cytokines”. Therefore, a preferred aspect of theinvention relates to an artificial antigen presenting cell (aAPC)scaffold comprising a polymeric backbone to which are attached thefollowing template molecules:

-   -   i. at least two different gamma-chain receptor cytokines, such        as at least two different gamma-chain receptor cytokines        selected from the group consisting of IL-21, IL-2, IL-15, IL-4,        IL-9 and IL-7,    -   ii. at least one antigen,    -   iii. optionally, at least one co-stimulatory molecule selected        from the group consisting of B7.2 (CD86), B7.1 (CD80), CD40,        ICOS and PD-L1, and    -   iv. optionally, at least one CD47 molecule.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the group of gamma-chain receptor cytokinesconsist of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the gamma-chain receptor cytokinesare selected from the group consisting of IL-21, IL-2, IL-15, IL-4, IL-9and IL-7.

The inventors have identified preferred combinations of stimulatorymolecules within the gamma-chain receptor cytokine family.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the gamma-chain receptor cytokinesare selected from the group consisting of IL-21, IL-2 and IL-15.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the gamma-chain receptor cytokines compriseat least IL-21.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the gamma-chain receptor cytokinescomprise:

-   -   i. at least IL-2 and IL-21, or    -   ii. at least IL-15 and IL-21.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the gamma-chain receptor cytokinesare:

-   -   i. IL-2 and IL-21, or    -   ii. IL-15 and IL-21.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the gamma-chain receptor cytokinescomprise:

-   -   i. at least IL-4 and IL-21,    -   ii. at least IL-7 and IL-21, or    -   iii. at least IL-9 and IL-21.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the gamma-chain receptor cytokinesare:

-   -   i. IL-4 and IL-21,    -   ii. IL-7 and IL-21, or    -   iii. IL-9 and IL-21.

The antigen may be a MHC presented antigenic peptide or an antigen thatis recognized without being bound to the MHC complex (i.e. non-MHCpresented molecule). Antigens that are not bound to a MHC complex may beany type of protein that is capable of inducing an immune response.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the at least one antigen is anon-MHC presented molecule.

A relevant class of non-MHC presented antigens are cluster ofdifferentiation (CD) proteins. CD proteins are a group of cell surfacemolecules commonly recognized as targets for cellular immunophenotypingand may act as receptor or ligands in signal cascades of importance tocell signaling. CD proteins may be included in aAPCs specificallydesigned to stimulate and expand chimeric antigen receptor (CAR) Tcells. Examples of relevant CD proteins include, but is not limited to,CD19, CD20, CD22 and CD269.

Another relevant class of antigens are haptens or organic smallmolecules, such as, but not limited to, biotin, fluorescein,digoxigenin, dinitrophenol, cotinine, hydralazine and urushiol. Thehapten may be conjugated to a disease targeted antibody. CAR T platformsusing anti-hapten CAR T cells in combination with hapten-conjugatedanti-cancer antibodies has been proposed as a novel way to targetmultiple cancer-antigens using single CAR T cells.

An embodiment of the present invention relates to the aAPC scaffold asdescribed herein, wherein the antigen is a non-MHC presented moleculeselected from the group consisting of CD19, CD20, CD22, CD269, haptens,BCMA, epidermal growth factor receptor (EGFR), mesothelin (MSLN),variant III of the epidermal growth factor receptor (EGFRvIII), humanepidermal growth factor receptor-2 (HER2), carcinoembryonic antigen(CEA), and prostate-specific membrane antigen (PSMA).

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the non-MHC presented molecule isa CD protein.

CD proteins may be included in aAPCs specifically designed to stimulateand expand chimeric antigen receptor (CAR) T cells. Examples of relevantCD proteins include, but is not limited to, CD19, CD20, CD22 and CD269.

Therefore, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the CD protein is selected fromthe group consisting of CD19, CD20, CD22 and CD269.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the non-MHC presented molecule is a hapten.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the hapten is attached to anantibody.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the antigen is a major histocompatibilitycomplex molecule comprising an antigenic peptide (pMHC).

The template molecules may be attached to the polymeric backbone via theinteraction between coupling agents and affinity tags. Coupling agentsare located on the polymeric backbone of the aAPC scaffold and may beattached to the backbone by, but not limited to, hydrophobicinteractions, electrostatic interactions or covalent bonding. Whenpositioned on the polymeric backbone, the coupling agents provide aflexible template to which affinity-tagged template molecules may befixed in a modular fashion. Affinity tags are molecular species thatbind specifically to the coupling agent through, but not limited to,non-covalent interactions. By attaching an affinity tag to each templatemolecule, it is therefore easy to assemble a custom-built aAPC scaffold.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the template molecules areattached to the polymeric backbone via non-covalent interactions betweena coupling agent located on the polymeric backbone and an affinity tagon the template molecule.

Many known compatible pairs of affinity tags and couplings agents may beused with the present invention and include, but are not limited to,biotin/streptavidin, biotin/avidin, biotin/neutravidin,biotin/strep-tactin, poly-His/metal ion chelate, peptide/antibody,glutathione-S-transferase/glutathione, epitope/antibody, maltose bindingprotein/amylase and maltose binding protein/maltose. Other knowncompatible pairs of affinity tags and couplings agents may also be usedwith the present invention.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the coupling agent/affinity tag isselected from the group consisting of biotin/streptavidin,biotin/avidin, biotin/neutravidin, biotin/strep-tactin, poly-His/metalion chelate, peptide/antibody, glutathione-S-transferase/glutathione,epitope/antibody, maltose binding protein/amylase and maltose bindingprotein/maltose.

Another preferred embodiment of the present invention relates to theaAPC scaffold as described herein, wherein the coupling agent isstreptavidin and the affinity tag is biotin.

The polymeric backbone of the aAPC scaffold to which the templatemolecules are attached may also be based on a variety of differentmaterials. Thus, several types of types of backbones may be used withthe present invention, including, but not limited to, polysaccharides,synthetic polysaccharides, vinyl polymers, poly ethylene glycol, polypropylene glycol, derivatised cellulosics, strep-tactin andpoly-streptavidin. Polysaccharides may be dextran or different variantsof dextrans, such as carboxy methyl dextran, dextran polyaldehyde, andcyclodextrins. An example of a synthetic polysaccharide is e.g. ficoll.Vinyl polymers include, but are not limited to, poly(acrylic acid),poly(acrylamides), poly(acrylic esters), poly(methyl methacrylate),poly(maleic acid), poly(acrylamide), poly(methacrylic acid) andpoly(vinylalcohol). Polymeric backbones consisting of derivatisedcellulosics include, but are not limited to, derivatised cellulosicsincluding carboxymethyl cellulose, carboxymethyl hydroxyethyl celluloseand hydroxy-ethyl cellulose.

Additionally, there exist commercially available polymeric backbonesthat can serve as the basis for forming self-assembling aAPC scaffoldsaccording to the present invention. These polymeric backbones include,but are not limited to, the Streptamers from IBA GmbH and BeckmanCoulter, which are based on the Strep-tactin protein that oligomerizesto form a multimer capable of binding several biotinylated moleculessuch as biotinylated pMHC complexes, cytokines and co-stimulatorymolecules.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the polymeric backbone is selectedfrom the group consisting of polysaccharides, vinyl polymers, polyethylene glycol, poly propylene glycol, strep-tactin andpoly-streptavidin.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the polymeric backbone is a polysaccharide.

A further and preferred embodiment of the present invention relates tothe aAPC scaffold as described herein, wherein the polysaccharide isdextran.

The size of the polymeric backbone sets the physical limits to how manytemplate molecules that can be attached to each aAPC scaffold. The sizeof the polymeric backbone is given by its molecular weight.

Therefore, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the dextran has a molecular weightin the range of 50-3000 kDa, such as 100-2500 kDa, such as 250-2500 kDa.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the dextran has a molecular weight selectedfrom the group of consisting of 250 kDa, 270 kDa, 750 kDa, and 2000 kDa.

In addition to the number of molecules attached to each aAPC scaffold,another important parameter is the density with which the templatemolecules are distributed on the polymeric backbone. The density may bevaried by adjusting the ratio between all molecules comprised by theaAPC scaffold. Thus, an embodiment of the present invention relates tothe aAPC scaffold as described herein, wherein the ratio betweenpolymeric backbone:pMHC molecule:co-stimulatory molecule:cytokine isselected from the group consisting of 1:1:1:1, 1:2:1:1, 1:4:1:1,1:4:2:1, 1:4:2:2, 1:10:5:5, 1:4:4:4, 1:8:8:8, 1:10:10:10, 1:20:20:20,1:30:30:30, 1:40:40:40, 1:50:50:50, 1:50:10:10 or 1:50:20:20. Anotherembodiment of the present invention relates to the aAPC scaffold asdescribed herein, wherein the ratio between polymeric backbone:pMHCmolecule:cytokine 1:cytokine 2 is selected from the group consisting of1:1:1:1, 1:2:1:1, 1:4:1:1, 1:4:2:1, 1:4:2:2, 1:10:5:5, 1:4:4:4, 1:8:8:8,1:10:10:10, 1:20:20:20, 1:30:30:30, 1:40:40:40, 1:50:50:50, 1:50:10:10or 1:50:20:20.

Still another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between polymericbackbone:pMHC molecule:co-stimulatory molecule:cytokine 1:cytokine 2 isselected from the group consisting of 1:1:1:1:1, 1:2:1:1:1, 1:4:1:1:1,1:4:2:1:1, 1:4:2:2:2, 1:10:5:5:5, 1:4:4:4:4, 1:8:8:8:8, 1:10:10:10:10,1:20:20:20:20, 1:30:30:30:30, 1:40:40:40:40, 1:50:50:50:50,1:50:10:10:10 or 1:50:20:20:20.

The present invention may be suitable for expansion of T cells from avariety of subjects. Thus, an embodiment of the present inventionrelates to the aAPC scaffold as described herein, wherein the at leastone pMHC molecule is a vertebrate MHC molecule, such as a human, murine,rat, porcine, bovine or avian molecule.

Another preferred embodiment of the present invention relates to theaAPC scaffold as described herein, wherein the vertebrate MHC moleculeis a human molecule.

As described above, MHC molecules exist in several variants. MHCmolecules include, but are not limited to, MHC class I molecules, MHCclass II molecules, MHC class III molecules, MHC class I like moleculesand MHC class II like molecules. MHC class I like molecules include, butare not limited to, CD1a, CD1b, CD1c, CD1d, MICA, MICB, MR1, ULBP-I,ULBP-2, and ULBP-3. MHC class II like molecules include, but are notlimited to, HLA-DM, HLA-DO, I-A beta2, and I-E beta2.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the at least one pMHC molecule isselected from the group consisting of MHC class I molecules, MHC classII molecules, MHC class III molecules, CD1a, CD1b, CD1c, CD1d and MR1.

Another preferred embodiment of the present invention relates to theaAPC scaffold as described herein, wherein the at least one pMHCmolecule is a MHC class I molecule.

A further preferred embodiment of the present invention relates to theaAPC scaffold as described herein, wherein the at least one pMHCmolecule is a human MHC class I molecule. In humans, the majorhistocompatibility complex (MHC) is encoded by a gene complex called thehuman leukocyte antigen (HLA) complex. The HLAs corresponding to MHCclass I are called HLA-A, HLA-B and HLA-C.

The antigenic peptide presented by the pMHC molecule ultimately decideswhich type of T cells will be expanded by the aAPC scaffold—the conceptpreviously referred to as MHC restriction. The antigens used with thepresent invention may essentially come from any source. The antigenicsource may include, but is not limited to, a human, a virus, abacterium, a parasite, a plant, a fungus, or a tumor. Thus, anembodiment of the present invention relates to the aAPC scaffold asdescribed herein, wherein the antigenic peptide of the pMHC is derivedfrom a source selected from the group consisting of a human, a virus, abacterium, a parasite, a plant, a fungus, and a tumor.

One use of the aAPC scaffold of the present invention is in theexpansion of tumor-reactive T cells for use in adoptive cell transfer(ACT). The strength of the ACT strategy is that T cells are present exvivo in an environment that, contrary to the local tumor environment, isoptimal for efficient expansion of an antigen specific T cellpopulation.

Another potential use of the aAPC scaffold of the present invention isfor expansion of a T cell population specific for fighting certaininfections that typically arise in the wake of transplantation. Patientsreceiving transplants are typically subject to immunosuppressivetreatment to avoid graft rejection. In many cases, such treatment leavesthe patient vulnerable to aggressive viral strains causing severeinfections of the already weakened patient. The aAPC scaffold of thepresent invention is perfectly suited for efficient expansion of T cellsextracted from transplantation patients, with the aim of treating anysevere infections by the ACT strategy.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the antigenic peptide of the pMHCis a cancer-associated epitope or virus epitope.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the antigen comprises a cancer-associatedepitope or virus epitope.

An alternative embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the antigenic peptide of the pMHCis a neoepitope, such as a cancer neoepitope.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the cancer-associated epitope is a virusepitope associated with a virus-induced cancer.

The aAPC scaffold of the present invention functions with any antigenicpeptide that may be presented by the pMHC molecules attached to thepolymeric backbone. Some indications are preferred in the presentinvention.

Thus, a preferred embodiment of the present invention relates to theaAPC scaffold as described herein, wherein the virus epitope is from avirus selected from the group consisting of human papillomavirus (HPV),Merkel cell polyomavirus (MCV), cytomegalovirus (CMV), Epstein-Barrvirus (EBV), human T-lymphotropic virus (HTLV), hepatitis B virus (HBV),hepatitis C virus (HCV) and influenza virus.

To optimize the efficiency of each aAPC scaffold with regard to theaccuracy with which the aAPC scaffold is capable of expanding a single Tcell specificity, in one version of the present invention, each aAPCscaffold is only harbouring a single variant of pMHC molecule, i.e. onlyone peptide antigen is presented for each type of aAPC scaffold.

Therefore, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the pMHC molecules are identicaland present only a single variant of an antigenic peptide.

By displaying only a single antigenic peptide for each aAPC scaffold,competition between T cells of different specificities is limited to aminimum. If desired, several different scaffolds presenting differentpeptides may be pooled together and expanded simultaneously. Thesimultaneous expansion of T cells with a variety of differentspecificities is possible because competition between T cell is kept ata minimum due to the aAPC scaffold clustering all the template molecules(i.e. the pMHC, co-stimulatory molecules and cytokines) in closeproximity to each other. Consequently, the T cell population expanded byuse of the aAPC scaffolds of the present invention retain specificityand the pool of different specificities ensures the breadth of anyimmune response if re-introduced into a subject. This lattercharacteristic is clinically important to avoid immune escape variants.The breadth of the response may be tuned by deciding how many differentaAPC scaffolds are pooled together in a single expansion.

The polymeric backbone may comprise any number of pMHc molecules that isreasonable according to the size of the polymeric backbone. Therefore,an embodiment of the present invention relates to the aAPC scaffold asdescribed herein, wherein each polymeric backbone comprises at least 5pMHC molecules, such as at least 8, such as at least 10, such as atleast 20, such as at least 30, such as at least 40, such as at least 50or such as at least 100.

An alternative embodiment of the present invention relates to the aAPCscaffold as described herein, wherein each polymeric backbone comprisesat least 2 pMHC molecules, such as at least 3 or such as at least 4.

For some applications it may be practical to immobilized the aAPCscaffolds on a solid support, e.g. for certain types of analytics or forseparation of the aAPC scaffolds from the expanded T cell population.Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein said aAPC scaffold is immobilizedon a solid support.

Many variants of solid supports exist and may be selected according tothe application of the aAPC scaffold. Variants of solid support include,but are not limited to, beads, well plates, particles, micro arrays,membranes, filters, gels and chips. Thus, an embodiment of the presentinvention relates to the aAPC scaffold as described herein, wherein thesolid support is selected from the group consisting of beads, wellplates, particles, micro arrays and membranes.

The aAPC scaffold may be attached to the solid support by anyconventional means, such as by linkers, antibodies or the like.

A plethora of different template molecules exist and therefore amultiplicity of different aAPC scaffold can be assembled. The inventorshave found that certain combinations of template molecules yieldespecially efficient and preferred aAPC scaffolds.

Thus, an embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the template molecules comprise atleast IL-21.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the template molecules comprise at leastIL-15 and IL-21.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the template molecules comprise atleast B7.2 (CD86).

Still another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecule is B7.2 (CD86), and    -   iii. the cytokines are IL-15 and IL-21.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the gamma-chain receptor cytokines are IL-15 and IL-21, and    -   iii. the co-stimulatory molecule is B7.2 (CD86).

An even further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the gamma-chain receptor cytokines are IL-15 and IL-21,    -   iii. the antigen is a major histocompatibility complex molecule        comprising an antigenic peptide (pMHC), and    -   iv. the co-stimulatory molecule is B7.2 (CD86).

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the ratio between pMHC, IL-15, IL-21 andB7.2 (CD86) on the dextran backbone is 2:1:1:1.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between dextranbackbone, pMHC, IL-15, IL-21 and B7.2 (CD86) is 1:10:5:5:5.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the gamma-chain receptor cytokinesare IL-2, IL-15 and IL-21.

An even further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the gamma-chain receptor cytokines are IL-2, IL-15 and        IL-21, and    -   iii. the antigen is a major histocompatibility complex molecule        comprising an antigenic peptide (pMHC).

An embodiment of the present invention relates to the aAPC scaffold asdescribed herein, wherein the ratio between dextran backbone, pMHC,IL-2, IL-15 and IL-21 is 1:10:5:5:5.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the gamma-chain receptor cytokines are IL-2, IL-15 and        IL-21,    -   iii. the antigen is a major histocompatibility complex molecule        comprising an antigenic peptide (pMHC), and    -   iv. the co-stimulatory molecule is B7.2 (CD86).

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the ratio between dextran backbone, pMHC,IL-2, IL-15, IL-21 and B7.2 (CD86) is 1:10:5:5:5:5.

Still another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the template molecules comprise atleast IL-6 and IL-10.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecule is B7.2 (CD86), and    -   iii. the cytokines are IL-6 and IL-10.

A still further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between pMHC, IL-6,IL-10 and B7.2 (CD86) on the dextran backbone is 2:1:1:1.

An even further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between dextranbackbone, pMHC, IL-6, IL-10 and B7.2 (CD86) is 1:10:5:5:5.

An embodiment of the present invention relates to the aAPC scaffold asdescribed herein, wherein the gamma-chain receptor cytokines comprise atleast IL-2 and IL-21.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein

-   -   i. the polymeric backbone is dextran, and    -   ii. the cytokines are IL-2 and IL-21.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran, and    -   ii. the gamma-chain receptor cytokines are IL-2 and IL-21.

An even further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the gamma-chain receptor cytokines are IL-2 and IL-21, and    -   iii. the antigen is a major histocompatibility complex molecule        comprising an antigenic peptide (pMHC).

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between pMHC, IL-2 andIL-21 on the dextran backbone is 1:1:1.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the co-stimulatory molecules comprise atleast B7.2 (CD86).

Still another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between dextranbackbone, pMHC, IL-2 and IL-21 is 1:8:8:8.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the polymeric backbone comprisesat least IL-1 and PD-L1.

A still further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecules are B7.2 (CD86) and PD-L1, and    -   iii. the cytokine is IL-1.

An even further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein the ratio between pMHC, IL-1, B7.2(CD86) and PD-L1 on the dextran backbone is 2:1:1:1.

Another embodiment of the present invention relates to the aAPC scaffoldas described herein, wherein the ratio between dextran backbone, pMHC,IL-1, B7.2 (CD86) and PD-L1 is 1:10:5:5:5.

Yet another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecules are B7.2 (CD86) and ICOS, and    -   iii. the cytokine is IL-10.

Still another embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran, and    -   ii. the cytokines are IL-1 and IL-2.

A further embodiment of the present invention relates to the aAPCscaffold as described herein, wherein

-   -   i. the polymeric backbone is dextran, and    -   ii. the cytokines are IL-2 and IL-15.

A further embodiment of the present invention relates to an artificialantigen presenting cell (aAPC) scaffold comprising a polymeric backboneto which are attached the following template molecules:

-   -   i. at least one major histocompatibility complex molecule        comprising an antigenic peptide (pMHC), and    -   ii. B7.2 (CD86), ICOS and PD-L1.

The aAPC scaffolds of the present invention may be part of a kitsuitable for use by hospitals and laboratories. Such a kit may compriseone or more different aAPC scaffolds suitable for expanding T cells withdifferent specificities, as well as medium suitable for expanding Tcells extracted from a sample. The kit may also hold other compounds ormolecules necessary for the expansion of a T cell-containing sample.

The aAPC scaffolds of the present invention may be used as animmunotherapy for direct administration into a subject to aid the immunesystem of the subject. The aAPC may be administered either locally orsystemically via any route, such as intravenous, intraperitoneal,intramuscular, subcutaneous, transdermal or oral.

Method of T-Cell Expansion

By extracting immune-reactive T cells from a unhealthy subject,expanding the T cells ex vivo and re-introducing the expanded T cellpopulation into the subject, it is possible to overcome some of thechallenges of immune suppressive diseases that otherwise render theimmune system paralysed. However, although the extraction of T cellsfrom e.g. peripheral blood by apheresis procedures and subsequentre-introduction into the patient is unproblematic, the activation andexpansion of T cells of a given specificity remains a great challengewith the resulting T cell population often lacking sufficientdifferentiation and functional capacity.

The aAPC scaffold of the present invention is suitable for simultaneousin vitro stimulation and expansion of T cells and yields T cellpopulations with a high ratio of active T cells, high antigenspecificity of the T cells and high functionality of the T cells. Thus,a second aspect of the present invention relates to a method forsimultaneous in vitro stimulation and expansion of T cells, comprisingthe following steps:

-   -   i. providing a sample comprising T cells,    -   ii. contacting said sample with a solution comprising an aAPC        scaffold as described herein,    -   iii. stimulating and expanding T cells with specificity for said        aAPC scaffold in culture, and    -   iv. harvesting the T cells of step iii) from the culture to        obtain an expanded antigen-specific population of T cells.

The sample comprising the T cells is extracted from a subject andsubsequently put into a culture comprising the aAPC scaffold underconditions that allow growth of the T cells. Thus, it is to beunderstood that the expansion of the T cells is to be carried out in asolution or medium that in addition to the aAPC scaffold contains allthe necessary compounds and factors for cell proliferation. Thus, theculture in which the T cell expansion is carried out may containcompounds that inhibit growth of irrelevant cells or promote growth ofthe T cells, e.g. IL-2.

To enhance the quality of the expanded T cell population, the aAPCscaffold may be filtered by centrifugation through a molecular weightcut-off filter in order to remove all non-bound pMHC molecules prior tomixing of the aAPC with the sample. This is to avoid stimulation frompMHC molecules not conjugated to scaffolds, and to remove excesspeptide, cytokines, and co-stimulatory molecules to limit thestimulation of irrelevant T cell subsets. The same applies to antigensnot complexed to MHC molecules, which can also be removed bycentrifugation through a molecular weight cut-off filter.

Thus, an embodiment of the present invention relates to the method asdescribed herein, wherein the solution comprising an aAPC scaffold ofstep ii) has been filtered before contact with the sample.

Another embodiment of the present invention relates to the method asdescribed herein, wherein the solution comprising an aAPC scaffold isfiltered by centrifugation through molecular weight cut-off filters.

An advantage of the aAPC scaffolds of the present invention is that theyallow simultaneous expansion of different T cell specificities becausethe cross-reactivity when using multiple different aAPC scaffolds isreduced to a minimum as explained above. The method of the presentinvention is therefore also effective for samples containing a varietyof T cells with different specificities.

Therefore, an embodiment of the present invention relates to the methodas described herein, wherein said sample of step i) comprises T-cells ofat least 2 different specificities, such as at least 5 differentspecificities, such as at least 10 different specificities, such as atleast 15 different specificities, such as at least 20 differentspecificities, or such as at least 50 different specificities.

Another embodiment of the present invention relates to the method asdescribed herein, wherein said solution comprising an aAPC scaffoldcomprises at least 2 different aAPC scaffolds, such as at least 5different aAPC scaffolds, such as at least 10 different aAPC scaffolds,such as at least 15 different aAPC scaffolds, such as at least 20different aAPC scaffolds, or such as at least 20 different aAPCscaffolds.

Yet another embodiment of the present invention relates to the method asdescribed herein, wherein T-cells of at least 2 different specificitiesare stimulated and expanded in parallel in the same sample, such as atleast 5 different specificities, such as at least 10 differentspecificities, such as at least 15 different specificities, or such asat least 20 different specificities.

A further embodiment of the present invention relates to the method asdescribed herein, wherein the method comprises the following steps:

-   -   i. providing a sample comprising T cells with at least 5        different specificities,    -   ii. contacting said sample with a solution comprising at least 5        different aAPC scaffolds,    -   iii. parallel stimulation and expansion of said T cells with at        least 5 different specificities for said at least 5 different        aAPC scaffolds in culture, and    -   iv. harvesting the T cells of step iii) from the culture to        obtain an expanded antigen-specific population of T cells with        at least 5 different specificities.

The sample comprising the T cells to be expanded may originate from anysource, but is typically extracted from blood, a tissue or a body fluid.Thus, an embodiment of the present invention relates to the method asdescribed herein, wherein the sample is selected from the groupconsisting of peripheral blood mononuclear cells, tumors, tissue, bonemarrow, biopsies, serum, blood, plasma, saliva, lymph fluid, pleurafluid, cerospinal fluid and synovial fluid.

The sample comprising the T cells to be expanded according to the methoddescribed herein may also be selected from stem cells, TCRmodified/transfected cells, chimeric antigen receptor (CAR) T cells.

Thus, an embodiment of the present invention relates to the method asdescribed herein, wherein the sample comprises CAR T cells and the atleast one antigen is not presented by a MHC molecule.

Thus, an embodiment of the present invention relates to the method asdescribed herein, wherein the sample comprises CAR T cells and the atleast one antigen of the aAPC scaffold is a CD protein.

Another embodiment of the present invention relates to the method asdescribed herein, wherein the sample comprises CAR T cells and the atleast one antigen is selected from the group consisting of CD19, CD20and CD22.

The method of the present invention may be used to expand any T cellexpressing the TCR necessary for interaction with the pMHC molecule onthe aAPC scaffold. The T cells suitable for expansion by the method ofthe present invention therefore include, but are not limited to, CD8 Tcells, CD4 T cells, regulatory T cells, natural killer T (NKT) cells,alpha-beta T cells, gamma-delta T cells, innate mucosal-associatedinvariant T (MAIT) cells, and lymphokine-activated killer (LAK) cells.

Thus, an embodiment of the present invention relates to the method asdescribed herein, wherein the T cells are selected from the groupconsisting of CD8 T cells, CD4 T cells, regulatory T cells, naturalkiller T (NKT) cells, gamma-delta T cells and innate mucosal-associatedinvariant T (MAIT) cells.

Another embodiment of the present invention relates to the method asdescribed herein, wherein the T cells are selected from the groupconsisting of CAR T cells, CD8 T cells, CD4 T cells, regulatory T cells,natural killer T (NKT) cells, gamma-delta T cells and innatemucosal-associated invariant T (MAIT) cells

A preferred embodiment of the present invention relates to the method asdescribed herein, wherein the T cells are CD8 T cells.

Yet another embodiment of the present invention relates to the method asdescribed herein, wherein the T cells are CAR T cells

For the re-introduction of an expanded T cell population into a patientto be meaningful from a therapeutic perspective, it is necessary thatthe extracted T cells are expanded to a clinically relevant number.Expansion of T cells by the method of the present invention is on theorder of 100-3000 fold. The number of cells available beforere-introduction into a patient is feasible is in the range of 10⁹-10⁹cells per administration. Cells are administered in a volume of 20 mL to1 L depending on the route of administration.

Therefore, an embodiment of the present invention relates to the methodas described herein, wherein the T cells are expanded to a clinicallyrelevant number.

As described above for the aAPC scaffold, the pMHC molecules may presenta variety of antigenic peptides. The same considerations regarding thechoice of antigenic peptides apply for the method. Thus, an embodimentof the present invention relates to the method as described herein,wherein the antigenic peptide of the pMHC is a cancer-associated epitopeor virus epitope.

Another embodiment of the present invention relates to the method asdescribed herein, wherein the antigen comprises a cancer-associatedepitope or virus epitope.

Another embodiment of the present invention relates to the method asdescribed herein, wherein the cancer-associated epitope is a virusepitope associated with a virus-induced cancer.

Yet another embodiment of the present invention relates the method asdescribed, wherein the virus epitope is from a virus selected from thegroup consisting of human papillomavirus (HPV), Merkel cell polyomavirus(MCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), humanT-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis C virus(HCV) and influenza virus.

Use of the Expanded T-Cell Population

It is envisioned that the expanded T cell population obtained by themethod of the present invention can be used effectively in a treatmentregimen focusing on adoptive immunotherapy (or adoptive cell transfer).In such a treatment regimen, immune-reactive T cells from a subject inneed of treatment are extracted. The subject may be any mammal, such ashumans, cows, pigs, birds, dogs, cats, mice, rats and the like. Thesource of the T cells may for example be peripheral blood mononuclearcells, tumors, tissue, bone marrow, biopsies, serum, blood, plasma,saliva, lymph fluid, pleura fluid, cerospinal fluid or synovial fluid.

Once extracted from the subject, the sample containing the T cells ofthe desired specificity or specificities is expanded using an aAPCscaffold customized to the subject and the condition or the disease tobe treated. This expansion is conducted in accordance with the method ofthe present invention as described above. When the T cell population hasbeen expanded to a clinically relevant number, it is administered to thesubject to induce an immune response and treat the disease.

Consequently, a third aspect of the present invention relates to anexpanded T cell population obtained by the method as described herein.

A fourth aspect of the present invention relates to an expanded T-cellpopulation obtained by the method as described herein for use as amedicament.

More specifically, an embodiment of the of the present invention relatesto a method for adoptive immunotherapy of a disease or disordercomprising

-   -   i. extracting a sample comprising T cells from a subject,    -   ii. contacting said sample with a solution comprising an aAPC        scaffold as described herein,    -   iii. stimulating and expanding T cells with specificity for said        aAPC scaffold in culture,    -   iv. harvesting the T cells of step iii) from the culture to        obtain an expanded antigen-specific population of T cells, and    -   v. administering the expanded antigen-specific population of T        cells to the subject in an amount effective to induce an immune        response.

As described above for the aAPC scaffold, the pMHC molecules may presenta variety of antigenic peptides. The same considerations regarding thechoice of antigenic peptides apply for the use of the expanded T-cellpopulation obtained by the method of the present invention.

Thus, a fifth aspect of the present invention relates to an expandedT-cell population obtained by the method as described herein for use inthe treatment of a cancer or viral condition.

An embodiment of the present invention relates to the expanded T-cellpopulation for use as described herein, wherein the cancer is associatedwith a viral condition.

Another embodiment of the present invention relates to the expandedT-cell population for use as described herein, wherein the viralcondition is associated with a virus selected from the group consistingof human papillomavirus (HPV), Merkel cell polyomavirus (MCV),cytomegalovirus (CMV), Epstein-Barr virus (EBV), human T-lymphotropicvirus (HTLV), hepatitis B virus (HBV), hepatitis C virus (HCV) andinfluenza virus.

The expanded T cell population obtained by the method as describedherein may be formulated in a pharmaceutical composition furthercomprising one or more adjuvants and/or excipients and/or apharmaceutically acceptable carriers. The excipients may include, butare not limited to, buffers, suspending agents, dispersing agents,solubilising agents, pH-adjusting agents and/or preserving agents.

The pharmaceutical composition may be used in adoptive immunotherapy (oradoptive cell transfer) for administration either locally orsystemically via any route, such as intravenous, intraperitoneal,intramuscular, subcutaneous, transdermal or oral.

It should be noted that embodiments and features described in thecontext of one of the aspects of the present invention also apply to theother aspects of the invention.

All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the followingnon-limiting examples.

EXAMPLES Example 1: Assembly of Artificial Antigen Presenting (aAPC)Scaffolds and Specific T Cell Expansion Using the aAPC (FIG. 1)

Here is described how aAPC scaffolds can be made by coupling pMHCcomplexes, cytokines and stimulatory molecules to dextran via astreptavidin-biotin interaction. In principle, biotin-streptavidin canbe replaced by any dimerization domain, where one half of thedimerization domain is coupled to the pMHC complex, cytokine orco-stimulatory molecule and the other half is coupled to dextran orsimilar scaffold backbone (see FIG. 1A).

Streptavidin modified dextran is commercially available in various sizesof dextran such as MW 250 KDa, 750 KDa, 2000 KDa from Fina Biosolutionsand from Immudex with dextran of approximately 270 KDa. The pMHCmonomers can be produced by classical E. coli expression methods or itcan be bought commercially from suppliers such as BioLegend. pMHC,cytokines and co-stimulatory molecules can be biotinylated by bothstandard chemical and enzymatic protocols. For example, pMHC can beenzymatically biotinylated by including a biotinylation consensuspeptide sequence in the MHC heavy chain allowing site-specificbiotinylation using BirA enzyme and free biotin. Cytokines andco-stimulatory molecules are commercially available from suppliers suchas BioLegend and PreProtech. These proteins are readily biotinylated byusing commercially available biotinylation reagents such as EZ-LinkSulfo-NHS-LC-Biotin from ThermoFisher Scientific and reacting accordingto supplier's protocol.

All the above given components were assembled to aAPC scaffolds via thestreptavidin-biotin interaction. Briefly, molecules were combined inaqueous buffer, such as PBS, in relative stoichiometry according to theexamples described below to give a final concentration of 60 nMassembled aAPC scaffold. The aAPC scaffold was allowed to assemble at 4°C. for one hour and was thereafter kept at 4° C. until addition to thecell culture. Assembled scaffolds can be stored at 4° C. for at leastone month. Assembled aAPC scaffold can be purified and separated fromunbound peptide, pMHC, cytokines and co-stimulatory molecules bycentrifuging unbound molecules through a MW cut-off filter such as anAmicon Ultra centrifugal filter units Ultra-4, MWCO 100 kDa.

The T cell cultures were established from human PBMCs or TILs andinitiated with 2×10⁶ cells/ml in a 48 well flat bottom culture platesand cultured for 2 weeks at 37° C. and 5% CO₂. The cells were stimulatedtwice a week by adding 0.2 nM final aAPC scaffold in 1 mL fresh X-VIVO15 media supplemented with 5% heat inactivated human serum and 20 IU/mlrecombinant human IL-2. After 1 week of culturing, the cells weretransferred to a 24 well flat bottom culture plates, and once a week asample was taken from the cultures for MHC tetramer staining to trackthe expansion of antigen-specific CD8 T cells by flow cytometry.

Assembled aAPC may be utilized to expand specific T cell populationsextracted from patients (see FIG. 1B). The T cells may for instance beCD8 T cells, CD4 T cells, regulatory T cells or natural killer T (NKT)cells that can be expanded to a clinically relevant number andreintroduced into the patient for treatment of disease. The use of aAPCscaffolds for expansion of T cells ensures a high ratio of active Tcells, high antigen specificity of the T cells and high functionality ofthe T cells. By using several different aAPC scaffolds in a singlesample, it is possible to simultaneously expand differentantigen-specific T cells without competition between specificities,thereby retaining T cell specificity and a pool of differentspecificities ensuring a broad immune response.

Example 2: Determination of Scaffold to Molecule Ratio (FIGS. 2 and 3)

It is important that the interaction between the antigen presentingscaffold, referred to as MHC-scaffold in figure legends, and T cellreceptors (TCRs) is directed by the pMHC molecules, and not by thecytokines or co-stimulatory molecules, since all T cells have receptorsfor binding these molecules. It is therefore fundamental to have anoptimal number and density of pMHC molecules attached to the antigenpresenting scaffold to ensure that the interaction is governed by thespecific interaction between pMHC and TCR. To determine the most optimalcomposition of the antigen presenting scaffolds and the number of pMHCmolecules required for a TCR-pMHC driven interaction, different ratiosof pMHC, cytokines, and co-stimulatory molecules were conjugated toscaffold backbones and applied in staining of healthy donor peripheralblood mononuclear cells (PBMCs) and analysed by flow cytometry.

Antigen presenting scaffolds carrying virus peptide HLA-A1 CMV pp65 YSEwere assembled in different scaffold:pMHC ratios and co-attached withco-stimulation, and were applied in staining of PBMCs of one healthydonor with response against the CMV pp65 YSE peptide. The meanfluorescence intensity (MFI) of each staining was used to determine theoptimal number of pMHC to be conjugated to the scaffolds, and stainingindex (SI) was used as a measure of separation between positive andnegative events. MFI and SI values of antigen-specific CD8 T cellsdetected for each staining are shown in FIG. 2 .

Conclusion:

(FIG. 2A) The ratios 1:10 and 1:20 (scaffold:pMHC) were found to beoptimal as these show the highest MFI values, but TCR-pMHC interactionsare retained even at 1:5 ratio. (FIGS. 2B-C) From these stainings itcould be concluded that the antigen presenting scaffold compositions1:10:5:5 and 1:20:5:5 (scaffold:pMHC:B7-2:IL-15) were optimal, sincethese show the highest MFI and SI values compared to ratio of1:10:10:10.

Antigen presenting scaffold ratios of 1:15:5:5(scaffold:pMHC:B7-2:IL-15), 1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21)and 1:8:8:8 (scaffold:pMHC:IL-2:IL-21) were used in further expansionexperiments of virus-specific CD8 T cells from healthy donor PBMCs.

Two control stainings were conducted with scaffolds not carrying anypMHC molecules, but with either B7-2 or IL-15 in ratios of 1:30(scaffold:B7-2) and (scaffold:IL-15) respectively. This was carried outto investigate the level of B7-2 and/or IL-15 mediated binding of theantigen presenting scaffolds to T cells. Representative dot plots areshown for the two control stainings in FIG. 3 .

Conclusion:

(FIGS. 3A-B) Based on the two control stainings, the interaction betweenscaffolds and unspecific T cells populations is very limited, as nosignificant binding was observed for any of the two scaffolds.

Example 3: Expansion of Antigen-Specific CD8 T Cells Using AntigenPresenting Scaffold (FIG. 4)

HLA-A1 FLU BP-VSD specific CD8 T cells from a healthy donor wereexpanded in parallel in the presence of either antigen presentingscaffold with the ratio 1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21),free FLU BP-VSD peptide, IL-15, and IL-21 cytokines, or antigenpresenting scaffold with the ratio 1:10:5:5:5 carrying an irrelevantpeptide specificity in the MHC complex. All cultures were supplementedwith 20 IU/ML IL-2 and cultured for 2 weeks. The expansion of the HLA-A1FLU BP-VSD specific CD8 T cells were traced by tetramer staining once aweek. Representative dot plots are shown in FIG. 4 .

Conclusion:

(FIGS. 4A-D) This experiment demonstrates that it is feasible to expandantigen-specific CD8 T cells with low frequent baseline responses in apMHC directed manner, using antigen presenting scaffolds. When comparingof expansion of cells stimulated with peptide, IL-15 and IL-21 addedfreely in the culture media, and cells stimulated with antigenpresenting scaffolds, it is clear that cells stimulated with antigenpresenting scaffolds have expanded the most both in frequency and inabsolute number of specific CD8 T cell (see FIGS. 4E-F). Furthermore,antigen presenting scaffolds carrying irrelevant peptide MHC specificitywere not able to stimulate A1 FLU BP-VSD specific CD8 T cell expansion,demonstrating that the cells cannot benefit from the co-attachedcytokines and co-stimulatory molecules, without established pMHCdirected interaction.

Example 4: Filtering of Antigen Presenting Scaffolds Prior to Expansionof Antigen-Specific CD8 T Cells (FIGS. 5-7)

After assembly of the antigen presenting scaffolds, these were filteredusing centrifugation through a molecular weight cut-off filter in orderto remove all non-bound pMHC molecules to avoid stimulation from pMHCnot conjugated to scaffolds, and to remove excess peptide, cytokines,and co-stimulatory molecules to limit the stimulation of irrelevant Tcell subsets. A number of experiments were carried out in parallel, inorder to investigate and compare the effect of filtering the antigenpresenting scaffolds vs. not filtering the antigen presenting scaffoldsprior to use in stimulation of antigen-specific CD8 T cells. Expansionlevels of various differentiation markers were investigated for theexpanded CD8 T cells in order to determine their phenotype. In theseexperiments antigen presenting scaffolds with the ratio 1:15:5:5(scaffold:pMHC:B7-2:IL-15) were used. See FIGS. 5-7 .

Conclusion:

(FIGS. 5 A-D) Cells stimulated with the filtered antigen presentingscaffolds show highest MFI, compared to cells stimulated with theunfiltered antigen presenting scaffold and free peptide, indicating thatthe filtered antigen presenting scaffold preferentially stimulates Tcells with high affinity TCRs or initiates an up-regulation of TCRexpression on the population of T cells expanded. However, the expansionrate of filtered MHC scaffolds was slightly compromised compared tounfiltered MHC scaffolds. The use of filtered reagents provides optimalfunctional characteristics (as shown later). Using both filtered andunfiltered scaffolds provides better T expansion and superior functionalcharacteristics compared to stimulation with free peptide-cytokines.

(FIGS. 6A-B) The highest expression of CD28 was detected for the highaffinity binding population (black population) stimulated with thefiltered antigen presenting scaffolds compared to stimulation with theunfiltered antigen presenting scaffold (dark grey population).

(FIGS. 7A-D) Cells stimulated with filtered antigen presenting scaffoldclearly obtain a higher CD28 expression compared to cells stimulatedwith unfiltered antigen presenting scaffold, meaning that filtration ofantigen presenting scaffolds prior to T cell stimulation cultures canprovide a more robust phenotype for adoptive transfer purposes—with highlevel of CD28 expression being associated with enhanced survival andexpansion in vivo. There was no significant difference in CCR7 and CD57expression between filtered and unfiltered antigen presenting scaffolds.

Example 5: Expression of Differentiation and Co-Inhibitory Markers onAntigen-Specific CD8 T Cells Expanded with Antigen Presenting Scaffolds(FIGS. 8 and 9)

T cells are characterized by different surface molecules that determinetheir differential and functional status. The differentiation status canbe defined by the T cell phenotype. T cells are dynamically evolvingover time of a given antigen exposure, but may roughly be categorizedinto four different group. Naïve T cell, Effector T cell (TEM),late-stage effector T cells (TEMRA), and central memory T cells. Thenaïve pool is responsible for mounting a T cell response to previouslyunexperienced pathogens, whereas reinfection is often cleared bymounting a strong and fast T cell response among T CM or certain groupsfor TEM. These T cells are highly proliferative and very responsive toantigen stimulation. For adoptive transfer purposes, such a phenotype ispreferable. Thus, we aim for high CD28 expression and low CD57expression. Some level of CCR7 expression is preferable due to theirhoming capabilities.

Tim-3, LAG-3 and PD-1 are exhaustion/activation markers on T cells.These molecules function to regulate immune responses following T cellactivation. They serve as a natural mechanism to avoid excessive T cellactivation, but in the context of immune therapy, ideally thesemolecules should be minimally expressed on T cells to provide theoptimal capacity for T cell function and expansion in vivo. PD-1 seemsto be the most critical and dynamic marker among these. Blocking of PD-1signaling has been shown to provide dramatic T cell activation, andconsequently cancer rejection in patients.

The expression of differentiation and co-inhibitory markers of theexpanded CD8 T cells was investigated for cultures expanded withfiltered and unfiltered antigen presenting scaffold with ratio1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21) and compared with cellsexpanded with free peptide and cytokines. Likewise, cultures expandedwith filtered and unfiltered antigen presenting scaffold with ratio1:8:8:8 (scaffold:pMHC:IL-2:IL-21) was compared with antigen presentingscaffold 1:8 (scaffold:pMHC) plus free cytokines in the culture media.See FIGS. 8-9 .

Conclusion:

(FIGS. 8A-D) These stainings show that the A1 FLU BP-VSD specific CD8 Tcells, stimulated with the filtered antigen presenting scaffolds withratio 1:10:5:5:5 and 1:8:8:8 generated cells with higher CD28expression, compared to all other stimulations. No particular differencein CCR7 and CD57 expression was observed for cells stimulated with thetwo different antigen presenting scaffold including the ratios1:10:5:5:5 and 1:8:8:8.

(FIGS. 9A-D) These stainings show that cells stimulated with filteredand unfiltered antigen presenting scaffolds with composition 1:10:5:5:5and 1:8:8:8 had the lowest positive expression of PD-1, compared tocells stimulated with free peptide and cytokines, and 1:8 antigenpresenting scaffold and free cytokines. No particular difference inTim-3 or LAG-3 expression was observed for any of the stimulations.Furthermore, the filtered antigen presenting scaffolds were able tostimulate the highest frequency of PD-1 negative A1 FLU BP-VSD specificCD8 T cells, compared to cells stimulated with unfiltered antigenpresenting scaffolds or free peptide and cytokines.

Example 6: Functionality of Expanded Antigen-Specific CD8 T Cells (FIG.10)

In order to characterize the functional capacity of expandedantigen-specific CD8 T cells after antigen presenting scaffoldstimulation, cells were challenged with antigen, and stained withintracellular cytokine antibodies to detect the production of TNF-α, andIFN-γ, and surface expression of degranulation marker CD107a. Theexpression of CD107a is associated with cytotoxic activity and abilityto induce apoptosis in target cells. The production of TNF-α and IFN-γin response to antigen recognition is important for indirect cytotoxickilling. CD8 T cells that express all three markers simultaneously areinterpreted as having high killing capacity.

The functionality results shown in FIG. 10 represent T cell culturesexpanded with filtered and unfiltered antigen presenting scaffold withratio 1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21) and compared withcells expanded with free peptide and cytokines. Likewise, culturesexpanded with filtered and unfiltered antigen presenting scaffold withratio 1:8:8:8 (scaffold:pMHC:IL-2:IL-21) were compared with cellsexpanded with antigen presenting scaffold 1:8 (scaffold:pMHC) plus freecytokines in the culture media. Furthermore, it was investigated whetherIL-2 as a supplement in the culture media was essential to stimulate CD8T cells for expansion, or whether it could be excluded from the media,and attached onto the scaffold, in a ratio 1:8:8:8.

Conclusion:

(FIG. 10A) The frequency of triple positive CD8 T cells was higher forcultures stimulated with either filtered or unfiltered antigenpresenting scaffolds when compared to cells stimulated with free peptideand cytokines. This implies that the scaffold-based interaction isrequired to provide efficient T cells stimulation and activation ofmulti-functional T cells.

(FIG. 10B) These results showed that it was possible to exclude IL-2from the culture media and instead attached it onto the antigenpresenting scaffold, and still obtain multifunctional CD8 T cells(triple positive). Moreover, cells stimulated with filtered antigenpresenting scaffolds obtain the highest frequency of multifunctional(triple positive) CD8 T cells, when compared to cells stimulated withunfiltered antigen presenting scaffolds.

Example 7: pMHC Directed Stimulation (FIG. 11)

It was investigated whether the antigen presenting scaffolds directtheir stimulatory signals in a pMHC dependent manner or whetherantigen-specific CD8 T cells could also benefit from stimulation solelybased on interaction with the co-attached cytokines and co-stimulatorymolecules on the antigen presenting scaffold, without relevant peptidespecificity in the MHC molecules. To investigate this HLA-A1 FLU BP-VSDspecific CD8 T cells from a healthy donor were stimulated for 2 weekswith either antigen presenting scaffolds with the 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21) carrying HLA-A3 LTA ASF in the MHCcomplex as an irrelevant peptide specificity, or HLA-A1 FLU BP-VSD as arelevant peptide specificity. The CD8 T cells were then compared intheir ability to express CD107a and produce TNF-α and IFN-γ, uponchallenge with HLA-A1 FLU BP-VSD peptide. The experiment was carried outin duplicate. Representative dot plots are shown in FIG. 11 .

Conclusion:

(FIGS. 11A-B) From these analyses it was evident that antigen presentingscaffold stimulation is exclusively directed by the specificity of thepMHC molecules attached, and cells cannot benefit from stimulation ofthe co-attached cytokines and co-stimulatory molecules without specificrecognition and interaction with pMHC molecules.

Example 8: Use of Antigen Presenting Scaffolds to Expand MultipleAntigen-Specific CD8 T Cells Simultaneously (FIG. 12)

The antigen presenting scaffold system holds a potential advantage instimulation of multiple CD8 T cell specificities simultaneously withoutoccurrence of peptide-competition. Broad T cell responses are preferablefor clinical applications to avoid immune escape by loss-of-targetmechanisms. However, using traditional peptide-based stimulationstrategies, competition among the peptides will occur for binding to MHCclass I molecules to be presented to T cells. Presentation advantagesare given to peptides with the highest binding affinity and stability.It is consequently challenging to stimulate multiple T cell responsesequally well with free peptides. When using antigen presenting scaffoldsto stimulate multiple CD8 T cell specificities simultaneously, peptidecompetition is avoided as the peptides are already inserted into MHCmolecules and assembled onto scaffolds.

To demonstrate this property of the antigen presenting scaffolds, anexperiment was conducted where PMBCs from a healthy donor with 4 virusresponses (HLA-A2 EBV BRLF1 YVL, HLA-A2 CMV pp65 NLV, HLA-A2 EBV LMP2FLY, and HLA-A2 FLU MP 58-66 GIL) were used to investigate how manydifferent specificities could potentially be stimulated simultaneouslyin the same culture using antigen presenting scaffolds.

Five cultures were established from this donor, where 4 cultures wereused to expand the 4 responses individually, and one culture was used toexpand all 4 responses simultaneously. Thus, 4 different antigenpresenting scaffold were assembled, each carried 1 of the 4 virusspecificities, and these were added individually to culture 1-4, thusone specificity was expanded per culture. Then, in culture 5 all 4antigen presenting scaffolds were added simultaneously to expand 4 virusresponses at the same time, in the same culture. Briefly, the cells werestimulated twice a week by adding for each specificity 0.2 nM final aAPCscaffold in 1 mL of fresh X-VIVO 15 media supplemented with 5% heatinactivated human serum and 20 IU/ml recombinant human IL-2. After 1week of culturing, the cells were transferred to a 24 well flat bottomculture plate, and once a week a sample was taken from the cultures forMHC tetramer staining to track the expansion of antigen-specific CD8 Tcells by flow cytometry. The number of pMHC specific T cellscorresponding to the 4 virus responses was assessed by tetramer stainingafter 2 weeks expansion. The fold expansion was thereafter calculatedfor each specificity by dividing the absolute number of specific CD8 Tcells, obtained after 2 weeks expansion, with the absolute number ofspecific CD8 T cells from baseline. The fold expansion results are shownin FIG. 12 .

It is contemplated that both responses expanded individually using theaAPC scaffolds, and responses expanded simultaneously using the aAPCscaffolds are expanded more efficiently than the corresponding templatemolecules free in solution. Thus, the corresponding template moleculesin solution infer competition between specificities (also irrelevantspecificities) and consequently result in an expanded T cell populationof lesser antigen specificity and functionality of the T cells than a Tcell population expanded using the aAPC scaffolds. This scenario isespecially relevant for simultaneous expansion of several specificities.

Conclusion:

(FIG. 12 ) From this experiment it is clear that the highest foldexpansion of antigen-specific CD8 T cells is achieved when only onespecificity is stimulated in one culture (black bars). When mixingdifferent antigen presenting scaffolds to expand 4 specificitiessimultaneously, the fold expansion decreases. Nevertheless, it isdemonstrated that the antigen presenting scaffolds are capable ofexpanding multiple specificities simultaneously. In clinical situations,starting material is often at a minimum, and it is an advantage tostimulate many different specificities in a single sample.

Example 9: The Combination of Stimulatory Molecules on the AntigenPresenting Scaffold Determines the Effect (FIGS. 13-15)

Here it is demonstrated that the combination of molecules, cytokines andco-stimulatory molecules, is important for the result of an antigenpresenting scaffold-based T cell expansion. Specific combinations can beused to provide sufficient stimulation to T cells in order for the cellsto gain high killing functionality and maintain a young phenotype, whichare key features for T cells that are to be used in TIL based ACT, as itcorrelates with increased tumor regression.

Two experiments were carried out to investigate the functionality andexpression of CD28 and PD-1 markers of antigen-specific CD8 T cellsafter stimulation with various antigen presenting scaffolds, carrying acombination of molecules that are known to provide differentialimmunological effects. These stimulations were compared with a referenceantigen presenting scaffold, carrying a combination of molecules thatwas previously validated (see FIGS. 4-12 ), and known to providepositive stimulation to CD8 T cells.

Antigen presenting scaffolds with the ratio 1:10:5:5:5 and 1:8:8:8 wereused in these experiments where HLA-A2 EBV LMP2 CLG specific CD8 T cellsfrom a healthy donor were expanded for 2 weeks.

In the experiment using the antigen presenting scaffold with ratio1:10:5:5:5 (scaffold:pMHC:B7-2:molecule 1:molecule 2), 21 differentscaffolds were generated, all carrying the same pMHC molecule, and theB7-2 molecule in ratio 1:10:5 (scaffold:pMHC:B7-2), and additionalattachment of different combinations of PD-L1, ICOS, OX40L, CD5, IL-1IL-6, IL-10 in ratio 5:5 (molecule 1:molecule 2). The functionality ofthe HLA-A2 EBV LMP2 CLG specific CD8 T cells after expansion with these21 different antigen presenting scaffolds is shown in FIG. 13 . Thereference scaffold with the ratio 1:10:5:5:5(scaffold:pMHC:B7-2:IL-15:IL-21) is shown in duplicate.

Likewise, an alternative scaffold was assembled with ratio 1:8:8:8(scaffold:pMHC:IL-2:molecule 1). 7 different antigen presentingscaffolds were assembled carrying the same pMHC specificity and IL-2 inratio 1:8:8 (scaffold:pMHC:IL-2) and additional attachment of differentcombinations of PD-L1, ICOS, OX40L, CD5, IL-1 IL-6, IL-10 in ratio 8(molecule 1). The number of specific T cells and the functionality ofthe HLA-A2 EBV LMP2 CLG specific CD8 T cells after expansion with eachof the 7 different antigen presenting scaffold are shown in FIG. 14 .The reference scaffold with the ratio 1:8:8:8 (scaffold:pMHC:IL-2:IL-21)is shown in duplicate.

Conclusion

(FIGS. 13 and 14 ) These experiments demonstrate the relative potency ofvarious molecules on the antigen presenting scaffolds to providerelevant stimulation to T cells in order for the cells to expand anddevelop favorable functional and phenotypical characteristics,characterized by low expression of PD1, high expression of CD28, andmultifunctional cytokine response (TNF-α, IFN-γ and CD107a) upon antigenrecognition. The expression of all three functional markerssimultaneously (circle 3) is interpreted as multi-functionality,indicating that these cells have the highest killing capacity. Antigenpresenting scaffolds with the combination scaffold:pMHC:B7-2:IL-1:PD-L1and scaffold:pMHC:B7-2:IL-15:IL-21 (1:10:5:5:5) (see FIGS. 13A-B), andthe scaffold with the combination scaffold:pMHC:IL-2:IL-1 andscaffold:pMHC:IL-2:IL-21(1:8:8:8) (see FIGS. 14A-B), yield the highestnumber of antigen-specific CD8 T cells with expression of all threemarkers simultaneously.

Conclusion:

(FIG. 15 ) from these analyses, the relative expression of CD28 and PD1was determined. Antigen presenting scaffolds with the combinationscaffold:pMHC:B7-2:IL-10:IL-6 and scaffold:pMHC:B7-2:IL-15:IL-21(1:10:5:5:5) (see FIG. 15A), and scaffold with combinationscaffold:pMHC:IL-2:ICOS and scaffold:pMHC:IL-2:IL-21 (1:8:8:8) (see FIG.15B), yield the highest expression of CD28 among antigen-specific CD8 Tcells. Furthermore, antigen presenting scaffold with the combinationscaffold:pMHC:B7-2:ICOS:IL10 and scaffold:pMHC:B7-2:PD-L1:IL6(1:10:5:5:5) (see FIG. 15A), and scaffold with combinationscaffold:pMHC:IL-2:IL21 and scaffold:pMHC:IL-2:IL1 (1:8:8:8) (see FIG.15B), yield the lowest expression of PD-1 among antigen-specific CD8 Tcells. High expression of CD28 and low expression of PD1 are idealfeatures for cell populations for adoptive cell therapy.

The optimal scaffold for T cell stimulation should yield antigenspecific T cells with high multifunctional properties, good expansion(high absolute number of specific cells), high expression of CD28 andlow expression of PD1. The most promising scaffolds combining thesefeatures are: Combinations of pMHC with IL2, IL15, IL21, B7-2, ICOS,IL1.

Example 10: Different Length of Scaffold in Stimulation ofAntigen-Specific CD8 T Cells (FIG. 16)

The use of various lengths of scaffolds as a backbone in antigenpresenting scaffolds was investigated. Scaffolds of 250 kDa, 750 kDa,and 2000 kDa were used as a backbone in antigen presenting scaffolds ofratio 1:10:5:5:5 (scaffold:pMHC:B7-2:IL-15:IL-21), and applied instimulation of HLA-A2 EBV LMP2 CLG specific CD8 T cells from a healthydonor for two weeks. The frequency of specific expansion was detected bytetramer staining in order to compare the expansion potential for thethree scaffolds.

For all other experiments, a 250 kDa scaffold has been used. Byemploying a longer scaffold, more molecules can be attached, which mayresult in increased TCR-pMHC interaction and stimulation, and possiblyan enhanced phenotypic and functional outcome of the T cells can beachieved. Alternatively, the molecules can be distributed better on alonger scaffold, because there was more space between the molecules,which may result in an improved stimulation of the T cells.

Conclusion:

(FIG. 16 ) From this experiment it is shown that all tested dextranlengths can be used as a scaffold in the antigen presenting scaffolds,as specific expansion of antigen specific T cells directed to thescaffold bearing pMHC moiety was obtained for all for them. The 750 kDAscaffold yield the highest frequency of specific CD8 T cells.

Example 11: Parallel Expansion of Multiple Antigen Specificities fromOne Donor Sample in on Culture (FIG. 17A-B)

Antigen specific CD8 T cells from a healthy donor, with five virusresponses, were expanded in parallel essentially as described in example1, except multiple T cell specificities were expanded in parallel andthe frequency of antigen specific T cells from day 0 (baseline) andafter 14 days expansion with aAPC scaffold 1:8:8:8(scaffold:pMHC:IL-2:IL-21) was determined using MHC tetramers.

In FIG. 17A, two cultures were established to individually expand HLA-A2EBV LMP2 FLY and HLA-A2 CMV pp65 NLV specific CD8 T cells, respectively,essentially as done in example 1. These two cultures were respectivelycompared to two cultures where HLA-A2 EBV LMP2 FLY and HLA-A2 CMV pp65NLV specific CD8 T cells were also individually expanded, respectively,though using 1/10 of the normal aAPC scaffold concentration plus 9/10concentration of aAPC scaffolds with non-matching HLA-types.

In FIG. 17B, material from one healthy donor containing five known virusresponses was used as the starting material to simultaneously expand allfive antigen specificities in one single culture. This was essentiallydone as in example 1 using 1/10 of the aAPC scaffold 1:8:8:8(scaffold:pMHC:IL-2:IL-21) concentration used in example 1 of the fiveantigen specificities respectively, plus 5/10 of aAPC scaffolds withnon-matching HLA-type. The frequency of antigen specific T cells foreach of the five specificities was measured at day 0 (baseline) andafter 14 days expansion using MHC tetramers. The specificity of the fivevirus responses are respectively, HLA-A2 FLU MP 58-66 GIL, HLA-A2 EBVLMP2 FLY, HLA-A2 CMV pp65 NLV, HLA-A2 EBV BRLF1 YVL, and HLA-A2 CMV IE1VLE.

Conclusion:

(FIG. 17A-B) From example 11 it can be concluded that antigen-specific Tcells (HLA-A2 EBV LMP2 FLY and HLA-A2 CMV pp65 NLV specific CD8 T cells)can be efficiently expanded when using 1/10 of the aAPC scaffoldconcentration used in example 1, plus 9/10 of aAPC scaffolds withirrelevant non-matching HLA-type (FIG. 17A). It can also be concludedthat five different virus responses from a single donor material can beefficiently expanded in parallel in a single culture by applyingmultiple aAPC scaffold specificities simultaneously (FIG. 17B).

Example 12: Expansion of Antigen-Specific T Cells with aAPC Scaffolds ofDifferent Size and Stoichiometry of Scaffold, pMHC and Cytokines (FIG.18A-B)

Antigen-specific CD8 T cells from a healthy donor were expanded in sixparallel cultures essentially as in example 1 and the frequency of Tcells with the given antigen specificity was measured after 2 weeksexpansion using MHC multimers. Scaffolds of MW 250 KDa, 750 KDa, and2000 KDa each tested with two different scaffold to molecule ratios wereused. (A) aAPC scaffold 1:8:8:8 (scaffold:pMHC:IL-2:IL-21), and (B) aAPCscaffold 1:24:24:24 (scaffold:pMHC:IL-2:IL-21).

Conclusion:

(FIG. 18A-B) From example 12 it can be concluded that antigen-specificCD8 T cells can be efficiently expanded with scaffolds of different MW,such as 250 KDa, 750 KDa, and 2000 KDa and scaffold:pMHC:IL-2:IL-21ratios of 1:8:8:8 (FIG. 18A) and 1:24:24:24 (FIG. 18B) for all scaffoldsizes.

Example 13: In Vivo Expansion of OVA-Specific CD8 T Cells in C57BL/6Mice Using aAPC Scaffolds (FIG. 19)

Four C57BL/6 mice were vaccinated with ovalbumin (OVA) and polyIC toestablish an antigen-specific T cell response restricted towards theC57BL/6 allele H2-Kb presenting the OVA derived peptide SIINFEKL (SEQ IDNO:1). The frequency of OVA-specific CD8 T cells were measured prevaccination, on day 7 and day 19 after i.p. vaccination with OVA+poly ICand day 7 after booster (day 28) using H2-Kb/SIINFEKL tetramers. Fourdifferent boosters were administrated on day 21 post vaccination. Mouse1 had PBS i.v., mouse 2 had OVA i.p., mouse 3 had aAPC scaffold 1:8:8:8with the H2-Kb/SIINFEKL (scaffold:pMHC:IL-2:IL21) i.v., and mouse 4 hadH2-Kb/SIINFEKL in the same concentration as assembled on the aAPCscaffold 1:8:8:8 i.v. (i.e. the booster for mouse 3). I.e. in mouse 4,the antigenic peptide was given as part of a pMHC complex, but withoutthe aAPC scaffold.

Conclusion:

(FIG. 19 ) Example 13 demonstrates that aAPC scaffolds can be applied invivo, that such application is safe (no toxicities observed) and theaAPCs efficiently expand an antigen-specific T cell response in vivo(FIG. 19 ).

Example 14: Comparison of Expansion of Antigen-Specific CD8 T CellsUsing aAPC Scaffold Versus Peptide Pulsed Monocyte Derived DendriticCells (FIG. 20A-D)

Dendritic cells were generated from autologous PBMC's from an HLA-A0201CMV positive donor using PromoCell's Dendritic Cell Generation protocoland Media, which promote in vitro maturation of human Monocytes (hMo)into mature CD83+ monocyte-derived Dendritic Cells (moDCs). Monocyteswere differentiated into moDCs, using a combination of PromoCellMonocyte Attachment Medium (C-28051) and the PromoCell Dendritic CellGeneration Medium (C-28050). Briefly, PBMC's in PromoCell MonocyteAttachment Medium were plated out in tissue culture plates at a densityof 2-3 million/cm2 for 1 hour at 5% CO2 and 37° C. Monocytes werecapture by removing non-adherent cells. Differentiation into immaturemoDC (day 0) was started by adding PromoCell Dendritic Cell GenerationMedium supplemented with 1× Component A of the Cytokine Pack moDC(supplied at 100×) and incubation for 3 days at 37° C. and 5% CO2.Medium change was performed on day 3 by aspirating the medium from thecells and adding fresh PromoCell DC Generation Medium supplemented with1× Component A of the Cytokine Pack moDC to the cells. The moDCmaturation process was completed by supplementing the whole volume with1× of Component B of the Cytokine Pack moDC (supplied at 100×) on day 6and incubated at 37° C. and 5% CO2 for an additional 40 hours. moDC'swere counted by Trypan Blue staining.

Peptide pulsed moDC's were generated by suspending 37.500 cells/mL inX-VIVO media containing 50 μg/ml peptide (CMV pp65 NLVPMVATV (SEQ IDNO:2)) and incubated for 4 hours at 37° C. After incubation, cells werewashed once in X-VIVO media and suspended in 50 μL X-VIVO+5%.

HLA-A201/CMV pp65 NLVPMVATV peptide specific T cells were expanded from100.000 PBMC's from a healthy donor with initially 0.01% HLA-A201/CMVpp65 NLVPMVATV positive T cells. The expansion was done under fourconditions in parallel in the presence of either (A) free MHC complex(HLA-A201/NLVPMVATV) and IL2 and IL21, (B) aAPC scaffold with the ratio1:8:8:8 (HLA-A201/NLVPMVATV) (scaffold:pMHC:IL-2:IL-21), (C) 37.500unpulsed moDC's derived from the same donor supplemented with IL-7 to afinal concentration of 120 U/ml (day one) and IL-12 to a finalconcentration of 120 U/ml (day 2) or (D) 37.500 NLVPMVATV peptide pulsedmoDC's derived from the same donor supplemented with IL-7 to a finalconcentration of 120 U/ml (day one) and IL-12 to a final concentrationof 120 U/ml (day 2). All conditions were cultured for 2 weeks in X-VIVOmedia supplemented with 5% human serum. The expansion of HLA-A201/CMVpp65 NLVPMVATV peptide specific T cells was traced by MHC tetramerstaining after two weeks. Representative dot plots are shown in FIG. 20.

Conclusion:

(FIG. 20 ) Example 14 demonstrates that expansion of antigen-specificCD8 T cells using aAPC scaffold is significantly (approximately a factorof 2) more efficient (FIG. 20B, 5.5%, 550 fold expansion) for expansionof antigen-specific T cells as compared to using peptide pulsed moDC(FIG. 20D, 2.8%, 280 fold expansion).

REFERENCES

-   WO2002072631-   WO2009003492-   WO2009094273    Items

The invention will now be described in further details in the followingnon-limiting items.

Item 1. An artificial antigen presenting cell (aAPC) scaffold comprisinga polymeric backbone to which are attached the following templatemolecules:

-   -   i. at least one major histocompatibility complex molecule        comprising an antigenic peptide (pMHC),    -   ii. at least one cytokine selected from the group consisting of        IL-21, IL-2, IL-15, IL-1, IL-6, IL-10 and IL-7,    -   iii. optionally, at least one co-stimulatory molecule selected        from the group consisting of B7.2 (CD86), B7.1 (CD80), CD40,        ICOS and PD-L1, and    -   iv. optionally, at least one CD47 molecule.

Item 2. The aAPC scaffold according to item 1, wherein the templatemolecules comprise at least two different cytokines selected from thegroup consisting of IL-21, IL-2, IL-15, IL-1, IL-6, IL-10 and IL-7.

Item 3. The aAPC scaffold according to item 1 or 2, wherein the templatemolecules are attached to the polymeric backbone via non-covalentinteractions between a coupling agent located on the polymeric backboneand an affinity tag on the template molecule.

Item 4. The aAPC scaffold according to item 3, wherein the couplingagent is streptavidin and the affinity tag is biotin.

Item 5. The aAPC scaffold according to any one of the preceding items,wherein the polymeric backbone is selected from the group consisting ofpolysaccharides, vinyl polymers, poly ethylene glycol, poly propyleneglycol, strep-tactin and poly-streptavidin.

Item 6. The aAPC scaffold according to any one of the preceding items,wherein the polymeric backbone is a polysaccharide.

Item 7. The aAPC scaffold according to items 5 or 6, wherein thepolysaccharide is dextran.

Item 8. The aAPC scaffold according to item 7, wherein the dextran has amolecular weight in the range of 50-3000 kDa, such as 100-2500 kDa, suchas 250-2500 kDa.

Item 9. The aAPC scaffold according to items 7 or 8, wherein the dextranhas a molecular weight selected from the group of consisting of 250 kDa,270 kDa, 750 kDa, and 2000 kDa.

Item 10. The aAPC scaffold according to any one of the preceding items,wherein the at least one pMHC molecule is a vertebrate MHC molecule,such as a human, murine, rat, porcine, bovine or avian molecule.

Item 11. The aAPC scaffold according to item 10, wherein the vertebrateMHC molecule is a human molecule.

Item 12. The aAPC scaffold according to any one of the preceding items,wherein the at least one pMHC molecule is selected from the groupconsisting of MHC class I molecules, MHC class II molecules, MHC classIII molecules, CD1a, CD1b, CD1c, CD1d and MR1.

Item 13. The aAPC scaffold according to item 12, wherein the at leastone pMHC molecule is a MHC class I molecule.

Item 14. The aAPC scaffold according to any one of the preceding items,wherein the antigenic peptide of the pMHC is a cancer-associated epitopeor virus epitope.

Item 15. The aAPC scaffold according to item 14, wherein thecancer-associated epitope is a virus epitope associated with avirus-induced cancer.

Item 16. The aAPC scaffold according to item 14 or 15, wherein the virusepitope is from a virus selected from the group consisting of humanpapillomavirus (HPV), Merkel cell polyomavirus (MCV), cytomegalovirus(CMV), Epstein-Barr virus (EBV), human T-lymphotropic virus (HTLV),hepatitis B virus (HBV), hepatitis C virus (HCV) and influenza virus.

Item 17. The aAPC scaffold according to any one of the preceding items,wherein the pMHC molecules are identical and present only a singlevariant of an antigenic peptide.

Item 18. The aAPC scaffold according to any one of the preceding items,wherein each polymeric backbone comprises at least 5 pMHC molecules,such as at least 8, such as at least 10, such as at least 20, such as atleast 30, such as at least 40, such as at least 50 or such as at least100.

Item 19. The aAPC scaffold according to any one of the preceding items,wherein said aAPC scaffold is immobilized on a solid support.

Item 20. The aAPC scaffold according to item 19, wherein the solidsupport is selected from the group consisting of beads, well plates,particles, micro arrays and membranes.

Item 21. The aAPC scaffold according to any one of the preceding items,wherein the template molecules comprise at least IL-21.

Item 22. The aAPC scaffold according to any one of the preceding items,wherein the template molecules comprise at least IL-15 and IL-21.

Item 23. The aAPC scaffold according to any one of the preceding items,wherein the template molecules comprise at least B7.2 (CD86).

Item 24. The aAPC scaffold according to any one of the preceding items,wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecule is B7.2 (CD86), and    -   iii. the cytokines are IL-15 and IL-21.

Item 25. The aAPC scaffold according to item 24, wherein the ratiobetween pMHC, IL-15, IL-21 and B7.2 (CD86) on the dextran backbone is2:1:1:1.

Item 26. The aAPC scaffold according to any one of items 24 or 25,wherein the ratio between dextran backbone, pMHC, IL-15, IL-21 and B7.2(CD86) is 1:10:5:5:5.

Item 27. The aAPC scaffold according to any one of items 1-20, whereinthe template molecules comprise at least IL-6 and IL-10.

Item 28. The aAPC scaffold according to item 27, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecule is B7.2 (CD86), and    -   iii. the cytokines are IL-6 and IL-10.

Item 29. The aAPC scaffold according to item 28, wherein the ratiobetween pMHC, IL-6, IL-10 and B7.2 (CD86) on the dextran backbone is2:1:1:1.

Item 30. The aAPC scaffold according to any one of items 28 or 29,wherein the ratio between dextran backbone, pMHC, IL-6, IL-10 and B7.2(CD86) is 1:10:5:5:5.

Item 31. The aAPC scaffold according to any one of items 1-21, wherein

-   -   i. the polymeric backbone is dextran, and    -   ii. the cytokines are IL-2 and IL-21.

Item 32. The aAPC scaffold according to item 31, wherein the ratiobetween pMHC, IL-2 and IL-21 on the dextran backbone is 1:1:1.

Item 33. The aAPC scaffold according to any one of items 31 or 32,wherein the ratio between dextran backbone, pMHC, IL-2 and IL-21 is1:8:8:8.

Item 34. The aAPC scaffold according to item 1, wherein the polymericbackbone comprises at least IL-1 and PD-L1.

Item 35. The aAPC scaffold according to item 34, wherein

-   -   i. the polymeric backbone is dextran,    -   ii. the co-stimulatory molecules are B7.2 (CD86) and PD-L1, and    -   iii. the cytokine is IL-1.

Item 36. The aAPC scaffold according to item 35, wherein the ratiobetween pMHC, IL-1, B7.2 (CD86) and PD-L1 on the dextran backbone is2:1:1:1.

Item 37. The aAPC scaffold according to any one of items 35 or 36,wherein the ratio between dextran backbone, pMHC, IL-1, B7.2 (CD86) andPD-L1 is 1:10:5:5:5.

Item 38. A method for simultaneous in vitro stimulation and expansion ofT cells, comprising the following steps:

-   -   i. providing a sample comprising T cells,    -   ii. contacting said sample with a solution comprising an aAPC        scaffold according to any one of the preceding items,    -   iii. stimulating and expanding T cells with specificity for said        aAPC scaffold in culture, and    -   iv. harvesting the T cells of step iii) from the culture to        obtain an expanded antigen-specific population of T cells.

Item 39. The method according to item 38, wherein the solutioncomprising an aAPC scaffold of step ii) has been filtered before contactwith the sample.

Item 40. The method according to item 39, wherein the solutioncomprising an aAPC scaffold is filtered by centrifugation throughmolecular weight cut-off filters.

Item 41. The method according to any one of items 38-40, wherein saidsample of step i) comprises T-cells of at least 2 differentspecificities, such as at least 5 different specificities, such as atleast 10 different specificities, such as at least 15 differentspecificities, such as at least 20 different specificities, or such asat least 50 different specificities.

Item 42. The method according to any one of items 38-41, wherein saidsolution comprising an aAPC scaffold comprises at least 2 different aAPCscaffolds, such as at least 5 different aAPC scaffolds, such as at least10 different aAPC scaffolds, such as at least 15 different aAPCscaffolds, such as at least 20 different aAPC scaffolds, or such as atleast 20 different aAPC scaffolds.

Item 43. The method according to any one of items 38-42, wherein T-cellsof at least 2 different specificities are stimulated and expanded inparallel in the same sample, such as at least 5 different specificities,such as at least 10 different specificities, such as at least 15different specificities, or such as at least 20 different specificities.

Item 44. The method according to any one of items 38-43, wherein themethod comprises the following steps:

-   -   i. providing a sample comprising T cells with at least 5        different specificities,    -   ii. contacting said sample with a solution comprising at least 5        different aAPC scaffolds,    -   iii. parallel stimulation and expansion of said T cells with at        least 5 different specificities for said at least 5 different        aAPC scaffolds in culture, and    -   iv. harvesting the T cells of step iii) from the culture to        obtain an expanded antigen-specific population of T cells with        at least 5 different specificities.

Item 45. The method according to any one of items 38-44, wherein thesample is selected from the group consisting of peripheral bloodmononuclear cells, tumors, tissue, bone marrow, biopsies, serum, blood,plasma, saliva, lymph fluid, pleura fluid, cerospinal fluid and synovialfluid.

Item 46. The method according to any one of items 38-47, wherein the Tcells are selected from the group consisting of CD8 T cells, CD4 Tcells, regulatory T cells, natural killer T (NKT) cells, gamma-delta Tcells and innate mucosal-associated invariant T (MAIT) cells.

Item 47. The method according to any one of items 38-46, wherein the Tcells are CD8 T cells.

Item 48. The method according to any one of items 38-47, wherein the Tcells are expanded to a clinically relevant number.

Item 49. The method according to any one of items 38-48, wherein theantigenic peptide of the pMHC is a cancer-associated epitope or virusepitope.

Item 50. The method according to item 49, wherein the cancer-associatedepitope is a virus epitope associated with a virus-induced cancer.

Item 51. The method according to item 49 or 50, wherein the virusepitope is from a virus selected from the group consisting of humanpapillomavirus (HPV), Merkel cell polyomavirus (MCV), cytomegalovirus(CMV), Epstein-Barr virus (EBV), human T-lymphotropic virus (HTLV),hepatitis B virus (HBV), hepatitis C virus (HCV) and influenza virus.

Item 52. An expanded T cell population obtained by the method accordingto any one of items 38-51.

Item 53. An expanded T-cell population obtained by the method accordingto any one of items 38-51 for use as a medicament.

Item 54. An expanded T-cell population obtained by the method accordingto any one of items 38-51 for use in the treatment of a cancer or viralcondition.

Item 55. The expanded T-cell population for use according to item 54,wherein the cancer is associated with a viral condition.

Item 56. The expanded T-cell population for use according to item 54 or55, wherein the viral condition is associated with a virus selected fromthe group consisting of human papillomavirus (HPV), Merkel cellpolyomavirus (MCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV),human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis Cvirus (HCV) and influenza virus.

The invention claimed is:
 1. An artificial antigen presenting cell(aAPC) scaffold comprising a polymeric backbone to which are attachedthe following template molecules: i. at least two different gamma-chainreceptor cytokines selected from the group consisting of IL-21, IL-2,IL-15, IL-4, IL-9, and IL-7, and ii. at least one antigen, wherein thepolymeric backbone is soluble and non-magnetic and wherein the at leasttwo different gamma-chain receptor cytokines comprise at least IL-21. 2.The aAPC scaffold according to claim 1, wherein the at least twogamma-chain receptor cytokines are selected from the group consisting ofIL-21, IL-2, and IL-15.
 3. The aAPC scaffold according to claim 1,wherein the at least two gamma-chain receptor cytokines comprise: i. atleast IL-2 and IL-21, or ii. at least IL-15 and IL-21.
 4. The aAPCscaffold according to claim 1, wherein the at least one antigen is amajor histocompatibility complex molecule comprising an antigenicpeptide (pMHC).
 5. The aAPC scaffold according to claim 4, wherein eachpolymeric backbone comprises at least 5 pMHC molecules.
 6. The aAPCscaffold according to claim 4, wherein the antigen comprises acancer-associated epitope or virus epitope.
 7. The aAPC scaffoldaccording to claim 6, wherein the virus epitope is from a virus selectedfrom the group consisting of human papillomavirus (HPV), Merkel cellpolyomavirus (MCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV),human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis Cvirus (HCV) and influenza virus.
 8. The aAPC scaffold according to claim1, wherein the polymeric backbone is a polysaccharide.
 9. The aAPCscaffold according to claim 1, wherein: i. the polymeric backbone isdextran, ii. the gamma-chain receptor cytokines are IL-2 and IL-21, andiii. the antigen is a major histocompatibility complex moleculecomprising an antigenic peptide (pMHC).
 10. The aAPC scaffold accordingto claim 1, wherein the co-stimulatory molecules comprise at least B7.2(CD86).
 11. The aAPC scaffold according to claim 1, wherein the templatemolecules comprise at least one CD47 molecule.
 12. The aAPC scaffoldaccording to claim 1, wherein the template molecules are attached to thepolymeric backbone via non-covalent interactions between a couplingagent located on the polymeric backbone and an affinity tag on thetemplate molecule.
 13. A method for simultaneous in vitro stimulationand expansion of T cells, comprising: i. providing a sample comprising Tcells, ii. contacting said sample with a solution comprising an aAPCscaffold according to claim 1, iii. stimulating and expanding T cellswith specificity for said aAPC scaffold in culture, and iv. harvestingthe T cells of step iii) from the culture to obtain an expandedantigen-specific population of T cells.
 14. The method according toclaim 13, wherein T-cells of at least 2 different specificities arestimulated and expanded in parallel in the same sample.
 15. The methodaccording to claim 13, wherein the method comprises: i. providing asample comprising T cells with at least 5 different specificities, ii.contacting said sample with a solution comprising at least 5 differentaAPC scaffolds, iii. parallel stimulation and expansion of said T cellswith at least 5 different specificities for said at least 5 differentaAPC scaffolds in culture, and iv. harvesting the T cells of step iii)from the culture to obtain an expanded antigen-specific population of Tcells with at least 5 different specificities.
 16. The method accordingto claim 13, wherein the antigen comprises a cancer-associated epitopeor virus epitope.
 17. The aAPC scaffold according to claim 1, whereinthe following template molecules are further attached to the polymericbackbone: i. at least one co-stimulatory molecule selected from thegroup consisting of B7.2 (CD86), B7.1 (CD80), CD40, ICOS, PD-L1, and/orii. at least one CD47 molecule.
 18. The aAPC scaffold according to claim1, wherein a concentration of each of the gamma-chain receptor cytokinesis below about 4.8 nM.